Pyrrolidine-containing monomers and oligomers

ABSTRACT

The invention relates to pyrrolidine monomeric units and to oligomers which are joined via phosphate linkages, including phosphorothioate, phosphodiester and phosphoramidate linkages.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a 371 of PCT/US95/00356 filed Jan. 11, 1995 and acontinuation-in-part of U.S. application Ser. No. 08/180,134, filed Jan.11, 1994 now U.S. Pat. No. 5,519,134.

The contents of the foregoing patent application are incorporated hereinby reference.

FIELD OF THE INVENTION

This invention is directed to pyrrolidine monomeric units and tooligomers constructed from them. The oligomers can be synthesized tohave either random or predefined sequences of monomeric units and can bejoined via phosphate linkages, including phosphorothioate,phosphodiester and phosphoramidate linkages. Each of the monomeric unitscan include a chemical moiety thereon for binding of the oligomericstructures to proteins, nucleic acid, and other biological targets. Inpreferred embodiments, the compounds of the invention act as inhibitorsof enzymes such as phospholipase A₂ and are used for the treatment ofinflammatory diseases including atopic dermatitis and inflammatory boweldisease.

BACKGROUND OF THE INVENTION

Phospholipases A₂ (PLA₂) are a family of enzymes that hydrolyze the sn-2ester linkage of membrane phospholipids resulting in release of a freefatty acid and a lysophospholipid (see, Dennis, E. A., The Enzymes, Vol.16, pp. 307-353, Boyer, P. D., ed., Academic Press, New York, 1983).Elevated levels of type II PLA₂ are correlated with a number of humaninflammatory diseases. The PLA₂ -catalyzed reaction is the rate-limitingstep in the release of a number of pro-inflammatory mediators.Arachidonic acid, a fatty acid commonly linked at the sn-2 position,serves as a precursor to leukotrienes, prostaglandins, lipoxins andthromboxanes. The lysophospholipid can be a precursor toplatelet-activating factor. PLA₂ is regulated by pro-inflammatorycytokines and, thus, occupies a central position in the inflammatorycascade (see, e.g., Dennis, ibid.; Glaser, et al., TiPs Reviews 1992,14, 92; and Pruzanski, et al., Inflammation 1992, 16, 451).

All mammalian tissues evaluated thus far have exhibited PLA2 activity.At least three different types of PLA₂ are found in humans: pancreatic(type I), synovial fluid (type II) and cytosolic. Studies suggest thatadditional isoenzymes exist. Type I and type II, the secreted forms ofPLA₂, share strong similarity with phospholipases isolated from thevenom of snakes. The PLA₂ enzymes are important for normal functionsincluding digestion, cellular membrane remodeling and repair, and inmediation of the inflammatory response. Both cytosolic and type IIenzymes are of interest as therapeutic targets. Increased levels of thetype II PLA₂ are correlated with a variety of inflammatory disordersincluding rheumatoid arthritis, osteoarthritis, inflammatory boweldisease and septic shock, suggesting that inhibitors of this enzymewould have therapeutic utility. Additional support for a role of PLA₂ inpromoting the pathophysiology observed in certain chronic inflammatorydisorders was the observation that injection of type II PLA₂ into thefootpad of rats (Vishwanath, et al., Inflammation 1988, 12, 549) or intothe articular space of rabbits (Bomalaski, et al., J. Immunol. 199, 146,3904) produced an inflammatory response. When the protein was denaturedbefore injection, no inflammatory response was produced.

The type II PLA₂ enzyme from synovial fluid is a relatively smallmolecule (about 14 kD) and can be distinguished from type I enzymes(e.g., pancreatic) by the sequence and pattern of its disulfide bonds.Both types of enzymes require calcium for activity. The crystalstructures of secreted PLA₂ enzymes from venom and pancreatic PLA₂, withand without inhibitors, have been reported (Scott, et al., Science 1990,250, 1541). Recently, the crystal structure of PLA₂ from human synovialfluid has been solved (Wery, et al., Nature 1991, 352, 79). Thestructures clarify the role of calcium and amino acid residues incatalysis. The calcium acts as a Lewis acid to activate the scissileester carbonyl and bind the lipid, and a His-Asp side chain dyad acts asgeneral base catalyst to activate a water molecule nucleophile. This isconsistent with the absence of any acyl enzyme intermediates, and isalso comparable to the catalytic mechanism of serine proteases. Thecatalytic residues and the calcium ion are at the end of a deep cleft(ca. 14 Å) in the enzyme. The walls of this cleft contact thehydrocarbon portion of the phospholipid and are composed of hydrophobicand aromatic residues. The positively-charged amino-terminal helix issituated above the opening of the hydrophobic cleft. Several lines ofevidence suggest that the N-terminal portion is the interfacial bindingsite. (see, e.g., Achari, et al., Cold Spring Harbor Symp. Quant. Biol.1987, 52, 441; Cho, et al., J. Biol. Chem. 1988, 263, 11237; Yang, etal., Biochem. J. 1989, 262, 855; and Noel, et al., J. Am. Chem. Soc.1990, 112, 3704).

Much work has been reported in recent years on the study of themechanism and properties of PLA₂ -catalyzed hydrolysis of phospholipids.In in vitro assays, PLA₂ displays a lag phase during which the enzymeadsorbs to the substrate bilayer and a process called interfacialactivation occurs. This activation may involve desolvation of theenzyme/lipid interface or a change in the physical state of the lipidaround the cleft opening. The evidence favoring this hypothesis comesfrom studies revealing that rapid changes in PLA₂ activity occurconcurrently with changes in the fluorescence of a membrane probe(Burack, et al., Biochemistry 1993, 32, 583). This suggests that lipidrearrangement is occurring during the interfacial activation process.PLA₂ activity is maximal around the melting temperature of the lipid,where regions of gel and liquid-crystalline lipid coexist. This is alsoconsistent with the sensitivity of PLA₂ activity to temperature and tothe composition of the substrate, both of which can lead to structurallydistinct lipid arrangements separated by a boundary region. Fluorescencemicroscopy was used to simultaneously identify the physical state of thelipid and the position of the enzyme during catalysis (Grainger, et al.,FEBS Lett. 1989, 252, 73). These studies clearly show that PLA₂ bindsexclusively at the boundary region between liquid and solid phase lipid.

While the hydrolysis of the secondary ester bond of1,2-diacylglycerophospholipids catalyzed by the enzyme is relativelysimple, the mechanistic and kinetic picture is clouded by the complexityof the enzyme-substrate interaction. A remarkable characteristic of PLA₂is that maximal catalytic activity is observed on substrate that isaggregated (i.e., phospholipid above its critical micelleconcentration), while low levels of activity are observed on monomericsubstrate. As a result, competitive inhibitors of PLA₂ either have ahigh affinity for the active site of the enzyme before it binds to thesubstrate bilayer or partition into the membrane and compete for theactive site with the phospholipid substrate. Although a number ofinhibitors appear to show promising inhibition of PLA₂ in biochemicalassays (see, e.g., Yuan, et al., J. Am. Chem. Soc. 1987, 109, 8071;Lombardo, et al., J. Biol. Chem. 1985, 260, 7234; Washburn, et al., J.Biol. Chem. 1991, 266, 5042; Campbell, et al., J. Chem. Soc., Chem.Commun. 1988, 1560; and Davidson, et al., Biochem. Biophys. Res. Commun.1986, 137, 587), reports describing in vivo activity are limited (see,e.g., Miyake, et al., J. Pharmacol. Exp. Ther. 1992, 263, 1302).

Traditional structure activity relationship type drug discovery givesunambiguous products but yet requires the preparation of numerousindividual test candidates. The preparation of each structure requiressignificant amounts of time and resources. Another drug discoveryapproach, de novo design of active compounds based on high resolutionenzyme structures, generally has not been successful. Yet anotherapproach involves screening complex fermentation broths and plantextracts for a desired biological activity. The advantage of screeningmixtures from biological sources is that a large number of compounds canbe screened simultaneously, in some cases leading to the discovery ofnovel and complex natural products with activity that could not havebeen predicted otherwise. One disadvantage is that many differentsamples must be screened and numerous purifications must be carried outto identify the active component, which often is present only in traceamounts.

In order to maximize the advantages of each classical approach, newstrategies for combinatorial unrandomization have been developed byseveral groups. Selection techniques have been used with libraries ofpeptides (see, e.g., Geysen, et al., J. Immun. Meth. 1987, 102, 259;Houghten, et al., Nature 1991, 354, 84; and Owens, et al., Biochem.Biophys. Res. Commun. 1991, 181, 402) and nucleic acids (see, e.g.,Wyatt, et al., (in press) Proc. Natl. Acad. Sci. USA; and Ecker, et al.,Nucleic Acids Res. 1993, 21, 1853). These selection techniques involveiterative synthesis and screening of increasingly simplified subsets ofoligomers. In using these selection techniques, subsets are assayed foractivity in either cell-based assays, or for binding or inhibition ofpurified protein targets.

One technique, called SURF (Synthetic Unrandomization of RandomizedFragments; see, e.g., Ecker, et al., ibid., involves the synthesis ofsubsets of oligomers containing a known residue at one fixed monomerposition and equimolar mixtures of residues at all other positions. Fora library of oligomers four residues long containing three monomers (A,B, C), three subsets would be synthesized (NNAN, NNBN, NNCN, where Nrepresents equal incorporation of each of the three monomers). Eachsubset is then screened in a functional assay and the best subset isidentified (e.g., NNAN). A second set of libraries is synthesized andscreened, each containing the fixed residue from the previous round, anda second fixed residue (e.g. ANAN, BNAN, CNAN). Through successiverounds of screening and synthesis, a unique sequence with activity inthe assay can be identified.

OBJECTS OF THE INVENTION

It is an object of this invention to provide novel pyrrolidine monomericunits.

It is another object of the invention to provide novel pyrrolidinemonomeric units that can be incorporated into novel oligomericstructures.

It is a further object to provide novel pyrrolidine monomeric units thatcan be linked together via phosphorus-containing backbones.

It is still another object to provide novel pyrrolidine based oligomersthat include a diversity of functional moieties thereon for binding tobiological sites of interest.

BRIEF DESCRIPTION OF THE INVENTION

Compounds of the invention include monomeric compounds of structure I:##STR1## wherein: X is H, a phosphate group, an activated phosphategroup, an activated phosphite group, or a solid support;

Y is H or a hydroxyl protecting group;

Z is L₁, L₁ -G_(x), L₂, L₂ -G₂, NR₃ R₄, a nitrogen-containingheterocycle, a purine, a pyrimidine, a phosphate group, a polyethergroup, or a polyethylene glycol group;

L₁ is alkyl having 1 to about 20 carbon atoms, alkenyl having 2 to about20 carbon atoms, or alkynyl having 2 to about 20 carbon atoms;

L₂ is aryl having 6 to about 14 carbon atoms or aralkyl having 7 toabout 15 carbon atoms;

G₁ is halogen, OR₁, SR₂, NR₃ R₄, C(═NH)NR₃ R₄, NHC(═NH)NR₃ R₄, CH═O,C(═O)OR₅, CH(NR₃ R₄) (C(═O)OR₅), C(═O)NR₃ R₄, a metal coordinationgroup, or a phosphate group;

G₂ is halogen, OH, SH, SCH₃, or NR₃ R₄ ;

R₁ is H, alkyl having 1 to about 6 carbon atoms, or a hydroxylprotecting group;

R₂ is H, alkyl having 1 to about 6 carbon atoms, or a thiol protectinggroup;

R₃ and R.sub. are, independently, H, alkyl having 1 to about 6 carbonatoms, or an amine protecting group;

R₅ is H, alkyl having 1 to about 6 carbon atoms, or an acid protectinggroup;

Q is L₁, G₃, L₁ -G₃ or G₃ -L₁ -G₃ ;

G₃ is C(═O), C(═S), C(O)--O, C(O)--NH, C(S)--O, C(S)--NH or S(O)₂ ;

n is 0 or 1.

In preferred embodiments, Y is an acid labile hydroxyl blocking groupsuch as a trityl, methoxytrityl, dimethoxytrityl or trimethoxytritylgroup. X preferably is a phosphoramidite. In certain preferredembodiments, n is 1 and Q is carbonyl, thiocarbonyl, carboxy, acetyl orsuccinyl.

In one preferred group of compounds, Z includes a nitrogen-containingheterocycle such as an imidazole, pyrrole or carbazole ring. In afurther preferred group, Z includes a purine or a pyrimidine nucleobasesuch as adenine, guanine, cytosine, uridine or thymine. In anotherpreferred group of compounds, Z includes an unsubstituted oramine-substituted alkyl group, or an aryl group having 6 to about 20carbon atoms. In yet another preferred groups of compounds, Z includesfluorenylmethyl, phenyl, benzyl, alkyl-substituted benzyl, polyethyleneglycol, glutamyl, or NR₃ R₄ groups. Further compounds of the inventioninclude oligomeric compounds of structure II: ##STR2## wherein: X is H,a phosphate group, an activated phosphate group, an activated phosphitegroup, a solid support, a conjugate group, or an oligonucleotide;

Y is H, a hydroxyl protecting group, a conjugate group or anoligonucleotide;

E is O or S;

EE is 0⁻, or N(Y₀)T₀ ; _(Y) ₀ is H, or Q₂ !_(jj) --Z₂ ;

T₀ is Q₁ !_(kk) --Z₁, or together Y₀ and T₀ are joined in a nitrogenheterocycle;

Q₁ and Q₂ independently, are C₂ -C₁₀ alkyl, C₂ -C₁₀ alkenyl, C₂ -C₁₀alkynyl, C_(4-C) ₇ carbocylo alkyl or alkenyl, a heterocycle, an etherhaving 2 to 10 carbon atoms and 1 to 4 oxygen or sulfur atoms, apolyalkyl glycol, or C₇ -C₁₄ aralkyl;

jj and kk independently, are 0 or 1;

Z₁ and Z₂, independently, are H, C₁ -C₂ alkyl, C₂ -C₂₀ alkenyl, C₂ -C₂₀alkynyl, C₆ -C₁₄ aryl, C₇ -C₁₅ aralkyl, a halogen, CH═O, OR₁, SR₂, NR₃R₄, C(═NH) NR₃ R₃, CH(NR₃ R₄), NHC(═NH)NR₃ R₄, CH(NH₂)C(═O)OH, C(═O)NR₃R₄, C(═O)OR₅, a metal coordination group, a reporter group, anitrogen-containing heterocycle, a purine, a pyrimidine, a phosphategroup, a polyether group, or a polyethylene glycol group;

Z is L₁, L₁ -G₁, L₂, L₂ -G₂, NR₃ R₄, a nitrogen-containing heterocycle,a purine, a pyrimidine, a phosphate group, a polyether group, or apolyethylene glycol group;

L₁ is alkyl having 1 to about 20 carbon atoms, alkenyl having 2 to about20 carbon atoms, or alkynyl having 2 to about 20 carbon atoms;

L₂ is aryl having 6 to about 14 carbon atoms or aralkyl having 7 toabout 15 carbon atoms;

G₁ is halogen, OR₁, SR₂, NR₃ R₄, C(═NH)NR₃ R₄, NHC(═NH)NR₃ R₄, CH═O,C(═O)OR₅, CH(NR₃ R₄) (C(═O)OR₅), C(═O)NR₃ R₄, a metal coordinationgroup, or a phosphate group;

G₂ is halogen, OH, SH, SCH₃, or NR₃ R₄ ;

R₁ is H, alkyl having 1 to about 6 carbon atoms, or a hydroxylprotecting group;

R₂ is H, alkyl having 1 to about 6 carbon atoms, or a thiol protectinggroup;

R₃ and R₄ are, independently, H, alkyl having 1 to about 6 carbon atoms,or an amine protecting group;

R₅ is H, alkyl having 1 to about 6 carbon atoms, or an acid protectinggroup;

Q is L₁, G₃, L₁ -G₃ or G₃ -L₁ -G₃ ;

G₃ is C(═O), C(═S), C(O)--O, C(O)--NH, C(S)--O, C(S)--NH or S(O)₂ ;

n is 0 or 1; and

m is 1 to about 50, preferably 1 to about 25.

Further compounds of the invention include chimeric oligomeric compoundshaving a central region comprising a phosphodiester or aphosphorothioate oligodeoxynucleotide interspaced between flankingregions comprising the above-described monomeric or oligomericstructures.

The invention further includes processes for preparing randomizedoligomeric compounds including the steps of selecting a group ofmonomers as described above and covalently bonding at least two of themonomers of said group. In preferred processes, the Z moiety of at leastone monomer of said group is different from the Z moiety of anothermonomer of said group.

The compounds of the invention can be used as inhibitors of variousenzymes including phospholipase A₂ enzyme. As inhibitors ofphospholipase A₂, the compounds are useful for the treatment ofinflammatory diseases including atopic dermatitis and inflammatory boweldisease. The oligomeric compounds of the invention can be used indiagnostics since they are capable of specifically hybridizing tonucleic acids of interest in the etiology of diseases. The compounds ofthe invention also can be used as research probes and primers,especially for the study of enzyme biochemistry and protein-nucleic acidinteractions.

BRIEF DESCRIPTION OF THE DRAWINGS

The numerous objects and advantages of the present invention may bebetter understood by those skilled in the art by reference to theaccompanying figures, in which:

FIG. 1 describes synthetic processes for oligomeric compounds accordingto the invention.

DETAILED DESCRIPTION OF THE INVENTION

The monomeric compounds of the invention each include a pyrrolidinemoiety that, in turn, bears a number of functional groups. Certain ofthese groups are used to link adjacent pyrrolidine moieties and formoligomeric structures. Typically, the groups used to effect such linkageare primary and secondary hydroxyl groups. During oligomer synthesis,the primary hydroxyl group typically is blocked with a protecting groupand the secondary hydroxyl group is reacted with an activated phosphategroup such as a β-cyanoethyl phosphoramidate group. As used herein, theterm activated phosphate group is intended to denote a phosphate groupthat bears a chemical modification thereon to enhance its reactivitywith nucleophiles. Similarly, the term activated phosphite group denotesa phosphite group that bears a chemical modification to enhance itsreactivity with nucleophiles. Numerous such modifications are known inthe art.

The monomeric compounds of the invention preferably are covalently boundusing phosphate linkages. This permits coupling via either solutionphase or solid phase chemistries. Representative solution phasetechniques are described in U.S. Pat. No. 5,210,264, issued May 11, 1993and commonly assigned with this invention. Representative solid phasetechniques are those typically employed for DNA and RNA synthesisutilizing standard phosphoramidite chemistry. (see, e.g., Protocols ForOligonucleotides And Analogs, Agrawal, S., ed., Humana Press, Totowa,N.J., 1993.) A preferred synthetic solid phase synthesis utilizesphosphoramidites as activated phosphates. The phosphoramidites utilizeP^(III) chemistry. The intermediate phosphite compounds are subsequentlyoxidized to the p^(V) state using known methods. This allows forsynthesis of the preferred phosphodiester or phosphorothioate phosphatelinkages depending upon oxidation conditions selected. Other phosphatelinkages can also be generated. These include phosphorodithioates,phosphotriesters, alkyl phosphonates, phosphoroselenates andphosphoramidates.

An acid labile protecting group such as a member of the trityl familypreferably can be used for protection of the primary hydroxyl group. Thetrityl family includes at least trityl, monomethoxytrityl,dimethoxytrityl and trimethoxytrityl. The dimethoxytrityl group ispreferred and can be added by reacting the primary hydroxyl group with4, 4'-dimethoxytrityl chloride. Other hydroxyl protecting groups can beused, such as those described by Beaucage, et al., Tetrahedron 1992, 48,2223.

The pyrrolidine moieties bear functional groups in addition to thosethat form inter-pyrrolidine linkages. When the monomeric compounds arelinked together, these functional groups provide diverse properties("diversity") to the resulting oligomeric compounds. The functionalgroups include hydrogen-bond donors and acceptors, ionic moieties, polarmoieties, hydrophobic moieties, aromatic centers, and electron-donorsand acceptors. Together, the properties of the individual monomerscontribute to the uniqueness of the oligomers in which they are found.Thus, a library of such oligomers would have a myriad of properties,i.e., "diversity." Collectively, the properties of the individualmonomers that together form an oligomer contribute to the uniqueness ofsuch oligomer and impart certain characteristics thereto for interactionwith cellular, enzymatic or nucleic acid target sites.

In other aspects of the present invention the use of acid labile groupswhich are stable to the trichloroacetic acid treatment used for DMTremoval such as BOC-type protecting groups are used. They are stable toextended TCA treatment, but are removed by trifluoroacetic acidsolutions (e.g. 5% in CH₂ Cl₂). Another protecting group class which iscompatible to this methodology is the Allyl class. These groups arecleaved using transition metal catalysts. This type of protecting groupis particularly valuable in cases where the selective deprotection of aparticular functional group is desired while the oligomer is stillattached to the solid support, allowing a new reactive site to beuncovered. Additional protecting group tactics are possible: e.g.photolabile protecting groups are also compatible with this methodology.

Nitrogen heterocycles suitable for use as the functional group includeimidazole, pyrrole, pyrazole, indole, 1H-indazole, α-carboline,carbazole, phenothiazine, and phenoxazine groups. A more preferred groupof nitrogen heterocycles includes imidazole, pyrrole, and carbazolegroups. Imidazole groups are especially preferred.

Purines and pyrimidines according to the invention include adenine,guanine, cytosine, uridine, and thymine, as well as other synthetic andnatural nucleobases such as xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 5-halo uracil andcytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudo uracil),4-thiouracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other8-substituted adenines and guanines, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine. Further purinesand pyrimidines include those disclosed in U.S. Pat. No. 3,687,808,those disclosed in the Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons,1990, and those disclosed by Englisch, et al., Angewandte Chemie,International Edition 1991, 30, 613.

Alkyl, alkenyl, and alkynyl groups according to the invention includebut are not limited to substituted and unsubstituted straight chain,branch chain, and alicyclic hydrocarbons, including methyl, ethyl,propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl,dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl,octadecyl, nonadecyl, eicosyl and other higher carbon alkyl groups.Further examples include 2-methylpropyl, 2-methyl-4-ethylbutyl,2,4-diethylpropyl, 3-propylbutyl, 2,8-dibutyldecyl, 6,6-dimethyloctyl,6-propyl-6-butyloctyl, 2-methylbutyl, 2-methylpentyl, 3-methylpentyl,2-ethylhexyl and other branched chain groups, allyl, crotyl, propargyl,2-pentenyl and other unsaturated groups, cyclohexane, cyclopentane,adamantane as well as other alicyclic groups, 3-penten-2-one,3-methyl-2-butanol, 2-cyanooctyl, 3-methoxy-4-heptanal, 3-nitrobutyl,4-isopropoxydodecyl, 4-azido-2-nitrodecyl, 5-mercaptononyl,4-amino-1-pentenyl as well as other substituted groups.

Aryl groups according to the invention include but are not limited tosubstituted and unsubstituted aromatic hydrocarbyl groups such as phenyland naphthyl groups. Aralkyl groups include but are not limited togroups having both aryl and alkyl functionality, such as benzyl andxylyl groups.

Metal coordination groups according to the invention include but are notlimited to hydroxamic acids, catecholamide, acetylacetone,2,2'-bipyridine, 1,10-phenanthroline, diacetic acid,pyridine-2-carboxamide, isoalkyldiamine, thiocarbamato, oxalate, glycl,histidyl and terpyridyl. Other metal coordination groups are known, asfor example see Mellor, D. P., Chemistry of Chelation and ChelatingAgents in International Encyclopedia of Pharmacology and Therapeutics,Section 70, The Chelation of Heavy Metals, Levine, W. G. Ed., PergamonPress, Elmford, N.Y., 1979.

Solid supports according to the invention include controlled pore glass(CPG), oxalyl-controlled pore glass (see, e.g., Alul, et al., NucleicAcids Research 1991, 19, 1527), TentaGel Support--anaminopolyethyleneglycol derivatized support (see, e.g., Wright, et al.,Tetrahedron Letters 1993, 34, 3373) or Poros--a copolymer ofpolystyrene/divinylbenzene.

A number of substituent groups can be introduced into compounds of theinvention in a protected (blocked) form and subsequently de-protected toform a final, desired compound. In general, protecting groups renderchemical functionality inert to specific reaction conditions and can beappended to and removed from such functionality in a molecule withoutsubstantially damaging the remainder of the molecule. See, e.g., Greenand Wuts, Protective Groups in Organic Synthesis, 2d edition, John Wiley& Sons, New York, 1991. For example, amino groups can be protected asphthalimido groups or as 9-fluorenylmethoxycarbonyl (FMOC) groups andcarboxyl groups can be protected as fluorenylmethyl groups.Representative hydroxyl protecting groups are described by Beaucage, etal., Tetrahedron 1992, 48, 2223. Preferred hydroxyl protecting groupsare acid-labile, such as the trityl, monomethoxytrityl, dimethoxytrityl,and trimethoxytrityl groups.

Substituent groups according to the invention include but are notlimited to halogen (Cl, Br, F), hydroxyl (OH), thiol (SH), keto (C═O),carboxyl (COOH), ethers, thioethers, amidine (C(═NH)NR₃ R₄, guanidine(NHC(═NH)NR₃ R₄, glutamyl CH(NR₃ R₄)(C(═O)OR₅), nitrate (ONO₂), nitro(NO₂), nitrile (CN), trifluoromethyl (CF₃), trifluoromethoxy (OCF₃),O-alkyl, S-alkyl, NH-alkyl, N-dialkyl, O-aralkyl, S-aralkyl, NH-aralkyl,amino (NH₂), azido (N₃), hydrazino (NHNH₂), hydroxylamino (ONH₂),sulfoxide (SO), sulfone (SO₂), sulfide (S--), disulfide (S--S), silyl,heterocyclic, alicyclic and carbocyclic. Preferred substituents includehalogens, alcohols and ethers (OR₁), thiols and thioethers (SR₂), amines(NR₃ R₄), amidines C(═NH)NR₃ R₄ !, guanidines NHC(═NH)NR₃ R₄ !,aldehydes (CH═O), acids C(═O)OH!, esters C(═O)OR₅ !, amides C(═O) NR₃R_(4!) and glycine CH(NH₂)(C(═O)OH)!.

In preferred embodiments, Z includes an aminoethyl, carboxyethyl,adenylmethyl, thyminylmethyl, imidazolylmethyl, benzyl, 4-hexylbenzyl,myristyl, isopropyl, or tetraethylene glycol group. Z can be directlyattached to the pyrrolidine ring or can be attached via a tether, Q.Preferred tethers include alkyl and acyl (carbonyl-containing) groups.Preferred acyl groups include carbonyl, thiocarbonyl, carboxy, acetyl,and succinyl groups.

The compounds of the invention can include conjugate groups covalentlybound to primary or secondary hydroxyl groups. Conjugate groups of theinvention include intercalators, reporter molecules, polyamines,polyamides, polyethylene glycols, polyethers, groups that enhance thepharmacodynamic properties of oligomers, and groups that enhance thepharmacokinetic properties of oligomers. Typical conjugates groupsinclude cholesterols, phospholipids, biotin, phenanthroline, phenazine,phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines,coumarins, and dyes. Groups that enhance the pharmacodynamic properties,in the context of this invention, include groups that improve oligomeruptake, enhance oligomer resistance to degradation, and/or strengthensequence-specific hybridization with RNA. Groups that enhance thepharmacokinetic properties, in the context of this invention, includegroups that improve oligomer uptake, distribution, metabolism orexcretion. Representative conjugate groups are disclosed inInternational Patent Application PCT/US92/09196, filed Oct. 23, 1992,U.S. patent application Ser. No. 116,801, filed Sep. 3, 1993, and U.S.Pat. No. 5,218,105. Each of the foregoing is commonly assigned with thisapplication. The entire disclosure of each is incorporated herein byreference.

To synthesize a combinatorial library having a large degree of chemicaldiversity is an important aspect of the present invention. Chemicaldiversity is introduced at one level by varying the nature of thephosphorus likage. Phosphorus linkages amenable to the present inventioninclude phosphodiester (OPO), phosphorothioate (OPS), phosphoramidate(OPN), phosphorothioamidate (SPN), and alkylphosphonate (OPC). Thecombinatorial library can be prepared with a single type of phosphoruslinkage, or with different linkages at each position of the oligomer.For example, a single OPS linkage can be selectively introduced at anyposition in a OPO oligomer. In fact, all possible combinations of OPO,OPS, OPN, SPN and OPC linkages can be introduced selectively into theoligomers. The presence or absence of a type of linkage at a particularposition in an oligomer will have a profound effect on the properties ofthe molecule.

In the case of phosphoramidate linked libraries, a further level ofdiversity is possible due to the ability to have substituents on thenitrogen atom of the OPN linkage. Thus it is possible to introduce awide variety of amines each time a OPN linkage is present in anoligomer. It is possible to have the same amine substituents at each OPNlinkage or a different amine at each position.

Chemical diversity can be generated at several levels in SURF libraries.We have described below the preparation of a large number of monomers.These monomers have been prepared to explore two aspects of chemicaldiversity: first a wide number of functional groups are available,covering a range of chemical properties. Second, these functional groupscan be attached to different linker portions designed to display, orpresent them in space in different ways, allowing variable flexibility.The following section describes a third level of diversity, theinter-residue linkage itself. It is to be understood that the methodsused to introduce any of the linkage types are compatible. Specifically,by using the proper conditions, it is possible to introduce phosphatelinkages including OPO, OPS, OPN, SPN and OPC linkages at will at anyposition in an oligomer, independently of the sequence of the oligomer.This is possible because the chemical reaction which defines the natureof the linkage is separate from the attachment of the monomer to thereactive group on the solid support (whether linker or the previousmonomer). Specifically, when a phosphoramidite monomer is treated withtetrazole and added to the solid support which has a free hydroxylgroup, a phosphite triester is obtained. This phosphite triester canthen be treated with Iodine (formulated in the same way as for DNAsynthesis on an automated synthesizer), giving a phosphodiester afterammonia deprotection. Alternatively, the phosphite triester can betreated with benzodithiole-3-one-1,1-dioxide to give the correspondingphosphorothioate.

Hydrogen phosphonate chemistry has the advantage of allowing additionalchemical modifications to be introduced into oligomers. Oligonucleotidephosphodiesters and phosphorothioates have been prepared using thisapproach, (see Froehler, B. C., Matteucci, M. D. Tetrahedron Lett. 1986,27, 469-472), as well as oligonucleotide phosphoramidates (see Froehler,B. C. Tetrahedron Lett. 1986, 27, 5575-5579. Letsinger, R. L., Singman,C. N., Histand, G., Salunkhe, M. J. Am. Chem. Soc. 1988, 110, 4470-4471.The synthesis of oligomers containing both phosphodiesters andphosphoramidates was reported, as well as the use of phosphoramiditechemistry in conjunction with the synthesis of phosphoramidates (seeJung, P. M., Histand, G., Letsinger, R. L. Nucleosides & Nucleotides,1994, 13, 1597-1605). In this latter work, alternating phosphodiesterand phosphoramidate oligomers were prepared by coupling phosphoramiditesand H-Phosphonates to the growing oligomer, followed by the appropriateoxidation step. In general, however, all the examples described thus farhave incorporated the same amine substitution at all phosphoramidatelinkages in the oligomer. These studies have shown the feasibility ofusing the phosphoramidate bond as an additional site for theincorporation of diverse functional groups. A wide variety of amines canbe used in the oxidative step, and the monomers of the present inventionsupport the necessary chemistry. Thus, for the preparation ofcombinatorial libraries incorporating phosphoramidate linkages, themonomers of the present invention are converted to the correspondingH-Phosphonate monoesters. In one aspect of the present invention this isaccomplished using PCl₃ and imidazole as the phosphitylating reagent(see Garegg, P. J., Regberg, T., Stawinski, J., Stromberg, R. Chem. Scr.1986, 26, 59-62). These H-phosphonates monomers are oligomerized onsolid support by activation with pivaloyl chloride, adamantoyl chlorideor other appropriate activating agent. The intermediate H-Phosphonatediesters are oxidized to the phosphate diesters in high yields usingiodine in aqueous pyridine. This allowed for the comparison of thecoupling efficiency of the H-phosphonate and phosphoramidite methods.Essentially the same coupling efficiency is achieved with bothmethodologies. The H-phosphonate diesters are converted tophosphoramidates by the use of a 10% solution of the appropriate aminein pyridine/CCl₄ (1:1). Under these conditions, a H-phosphonate diesteris oxidized to a phosphoryl chloride via an Arbuzov reaction, followedby displacement of the chloride by a primary or secondary amine. Thesecond step has proven to be quite general, with a wide variety ofamines giving satisfactory yields. Moreover, the yield ofphosphoramidate is comparable to the yield of phosphodiester.

Several types of libraries are available through this methodology. Thesimplest kind is a library made from a set of monomers of the presentinvention (a set of 4 to 16 or more monomers is typically used) of 2 to10 or more monomer units in length, which is substituted at phosphoruswith a single amine. These libraries are prepared by split beadsynthesis, following the H-phosphonate synthesis protocol rather thanphosphoramidite chemistry. The intermediate H-phosphonate diesters areleft intact until the final step. At that point the oligomer librarypools are oxidized with CCl₄ /Pyridine containing 10% of the appropriateprimary or secondary amine. This has the result of converting all theinterresidue linkages to phosphoramidates. The library therefore iscomposed of all possible sequences of the monomers, separated intosubsets unique at a fixed position, linked together by a constantphosphoramidate linkage. It should be evident that the final propertiesof the library will be determined by the choice of amine used in theoxidation step. Thus, water solubility, pharmacokinetics andpharmacodynamics of the library components can be modulated by thechoice of amine. It is also possible to prepare oligomer libraries withmixed linkages by having an intermediate oxidation step (see Gryaznov,S. M., Sokolova, N. I. Tetrahedron Lett. 1990, 31, 3205-3208; Gryaznov,S. M., Potapov, V. K. Tetrahedron Lett. 1991, 32, 3715-3718; Farooqui,F., Sarin, P. S., Sun, D., Letsinger, R. L. Bioconjugate Chem., 1991, 2,422-426; Iso, Y., Yoneda, F., Ikeda, H., Tanaka, K., Fuji, K.Tetrahedron Lett. 1992, 33, 503-506). Thus, a portion of the oligomerlibrary is synthesized by H-phosphonate chemistry, which can be oxidizedwith (R₂ NH, CCl₄ /Py or S8, CS₂ /TEA or H₂ O, CCl₄ /Py), and a secondportion of the library synthesized and oxidized with a second set ofreagents. This creates a chimeric library, where a segment of the randomoligomers in each subset has a different linkage than the rest of themolecule. By extension of this methodology, it is possible toincorporate a different linkage at each position of the oligomer libraryby having a different oxidation step after each monomer coupling. Thelinkage can be combinatorialized by performing a separate oxidation on aportion of the H-phosphonate diester-linked solid support, followed bypooling of the subsets in the same way that the monomer positions arerandomized. Thus, each monomer and the linkage between them can berandomized by a split synthesis strategy.

Monomeric compounds of the invention can be used to prepare oligomericcompounds having either preselected sequences or sequences determinedvia combinatorial strategies. One useful combinatorial strategy is theabove-noted SURF strategy, which is disclosed and claimed in U.S. patentapplication Ser. No. 749,000, filed Aug. 23, 1991, and PCT ApplicationUS92/07121, filed Aug. 21, 1992, both of which are commonly assignedwith this application. The entire disclosure of these applications areherein incorporated by reference.

Illustrative of the SURF strategy is a 2'-O-methyl oligonucleotidelibrary (see, Ecker et. al., ibid.) shown in Table I, below. Table Idescribes the selection of a 2'-O-methyl oligonucleotide for binding toan RNA hairpin. The K_(D) 's, i.e., the binding constants, weredetermined by gel shift. "X" is used to indicate the position beingvaried and underlining is used to indicate positions that become fixedduring successive iterations of the SURF strategy.

                  TABLE I                                                         ______________________________________                                                   K.sub.D (mM)                                                       Subsets      X=A    X=C        X=G  X=T                                       ______________________________________                                        Round 1                                                                       NNNNXNNNN    22     10         >100 >100                                      Round 2                                                                       NNNNCNXNN    >10    4          >10  >10                                       Round 3                                                                       NNXNCNCNN    >10    0.5        >10  >10                                       Round 4                                                                       NNCXCNCNN    >10    0.15       >10  >10                                       Round 5                                                                       NNCCCXCNN    0.08   >1         0.4  >1                                        Round 6                                                                       NNCCCACXN    0.05   >0.5       0.08 >0.5                                      Round 7                                                                       NXCCCACAN    >0.1   >0.1       0.03 >0.1                                      Round 8                                                                       NGCCCACAX    0.05   0.02       0.05 0.04                                      Round 9                                                                       XGCCCACAC    0.03   0.05       0.02 0.01                                      ______________________________________                                    

This SURF strategy has not been used for libraries except those thatemploy naturally-occurring nucleotides as phosphodiesters orphosphorothioates as monomeric units. Other combinatorial strategieshave only been used for libraries that employ amino acids as monomericunits.

One aspect of the present invention is the inclusion of monomericcompounds having structure I in the above-described SURF strategy. Thefunctional groups appended to these monomeric compounds can beincorporated into the libraries while retaining the advantages ofautomated phosphoramidite oligomer synthesis. These functional groupscan effect interactions of the following types: hydrogen-bond donor andacceptor, ionic, polar, hydrophobic, aromatic, and electron donors andacceptors. Preferred functional groups include aminoethyl, carboxyethyl,adenylmethyl, thyminylmethyl, imidazolylmethyl, benzyl, myristyl,isopropyl, and tetraethylene glycol groups.

One advantage of the present invention is that the simple design ofmonomeric compounds of the inventions allows for combining rational drugdesign with screen mechanisms for thousands of compounds. This isachieved by using the compounds of the invention in a combinatorialtechniques such as the SURF strategies.

In one preferred embodiment, functional groups appended to the monomericcompounds of the invention are selected for their potential to interactwith, and preferably inhibit, the enzyme PLA₂. Thus, the compounds ofthe invention can be used for topical and/or systematic treatment ofinflammatory diseases including atopic dermatitis and inflammatory boweldisease. In selecting the functional groups, advantage can be taken ofPLA₂ 's preference for anionic vesicles over zwitterionic vesicles. Inselecting the backbone that bears these functional groups, furtheradvantage can be taken of fact that the natural substrate of PLA₂contains a phosphate group. Therefore, phosphodiester orphosphorothioate and other phosphate linked oligomers preferably areselected, providing a negatively charged compound for binding with thepositively charged interfacial binding site of PLA₂.

Certain compounds of the invention include aromatic functional groups tofacilitate binding to the cleft of the PLA₂ enzyme. (see, Oinuma, etal., J. Med. Chem. 1991, 34, 2260; Marki, et al., Agents Actions 1993,38, 202; and Tanaka, et al., J. Antibiotics 1992, 45, 1071). Benzyl and4-hexylbenzyl groups are preferred aromatic groups. The compounds of theinvention can further include hydrophobic functional groups such astetraethylene glycol groups. Since the PLA₂ enzyme has a hydrophobicchannel, hydrophobicity is believed to be an important property ofinhibitors of the enzyme.

In certain embodiments of the invention, hydroxy pyrrolidinephosphoramidite monomeric compounds having structure I are incorporatedinto libraries of oligomeric compounds and increasingly less complexsubsets of oligomers are identified in combinatorial screeningtechniques such as the above-described SURF technique by successiverounds of screens. In one preferred embodiment, a library of oligomericcompounds functionalized with aminoethyl, carboxyethyl, adenylmethyl,thyminylmethyl, tetraethylene glycol, imidazolylmethyl, benzyl,isopropyl, myristyl or 4-hexylbenzyl groups are prepared and assayed forinhibition of PLA₂ activity. The PLA₂ assay can be effected using acombinatorial screening strategy such as the SURF strategy. For thisassay, the oligomer libraries are screened for inhibition of human typeII PLA₂ enzymatic activity. Typically, these libraries contain about8000 different compounds. Successive iterations of the SURF technique iseffected to select unique oligomers from the library. The librariesadditionally can be screened in other in vitro assays to determinefurther mechanisms of inhibition.

Upon identification of oligomers in a first phase of screening, furthermodifications can be made to the contents of the oligomer libraries. Forexample, if a first iteration of screening results in an active compoundthat contains a benzyl group, then in subsequent iterations of thescreen this aromatic residue can then be varied using substituted benzylgroups. In this way, structural activity is identified in a stepwisemanner to define potent inhibitors of the enzymatic activity.

To maximize the identification of a tight binding oligomeric inhibitorof PLA₂ via a combinatorial approach, an array of functional groupstypically are included in a randomized library. The oligomers areassembled in a manner analogous to oligonucleotide synthesis by thecoupling of monomeric, phosphoramidate units wherein the normalnucleotide structure is replaced by more diverse chemical groups. Insome of the monomeric units, the nucleobases of nucleotides have beenretained. In other, the nucleobases are replaced with other functionalgroups selected to provide different ligand-ligand interactions thanthat provided by the nucleobases. The sugar moiety of a normalnucleotide is replaced by a hydroxy pyrrolidine unit, i.e. a prolinol,to form a unique prolinol-phosphate backbone. This methodology providesfor a convergent preparation of a large number of monomers bearing awide variety of functional groups. Where necessary, functional groupsare protected with base labile protecting groups to allow one-stepdeprotection of the oligomer upon completion of the synthesis.

As noted above, monomeric compounds having structure I can be linkedwith one another to form homopolymeric structures or they can be linkedwith nucleotides and/or other moieties to form heteropolymericstructures. For example, chimeric structures can be formed that includeone or more regions or "stretches" of the monomeric units of inventionjoined to one or more regions or "stretches" of naturally occurring orsynthetic oligonucleotides or to other synthetic or natural oligomericcompounds such as peptides, peptoids, peptide nucleic acids, oligoand/or polysaccharides. Further, oligomeric compounds having structureII can be incorporated into chimeric structures along with the compoundsdisclosed in the patent application entitled "Monomeric Diols AndPhosphate Linked Oligomers Formed Therefrom," bearing attorney docketISIS-0868 and the patent application entitled "Oligonucleotide MimicsHaving Nitrogen-Containing Linkages," bearing attorney docket ISIS-1014.The foregoing patent applications are filed concurrently with thisapplication, are commonly assigned, and are incorporated herein byreference.

In one embodiment of the invention, oligomeric compounds are synthesizedas shown in FIG. 1. This synthetic strategy emphasizes attachment ofwidely different functional groups to a rigid hydroxyprolinolintermediate. Each monomer unit contains a primary hydroxyl that isprotected as a dimethoxytrityl (DMT) ether and a secondary hydroxyl thatis converted to a cyanoethyl-diisopropylamino phosphoramidite.Trans-hydroxyproline 1 is protected as the fluorenylmethyl carbamate(Fmoc), and the carboxylate function is reduced using borane inrefluxing tetrahydrofuran (THF). Diol 3 is then selectively protected atthe primary hydroxyl as a DMT, and the Fmoc group of 4 is removed usingpiperidine in dimethylformamide (DMF). A substituted carboxylic acid isthen covalently linked to the nitrogen of intermediate 5 using standardpeptide coupling methods (see Bodansky, M., Principles of PeptideSynthesis, 1984, Springer-Verlag, Berlin). Functional groups (R) thatrequire protection are derivatized using base labile protecting groups.Monomers 6 are converted to the phosphoramidites 7 under standardconditions (see Oligonucleotide synthesis, a practical approach, Gait,M. J. Ed., 1984, IRL Press, Oxford). These phosphoramidites are thenoligomerized, either in predetermined sequences using standardoligonucleotide type synthetic procedures on a DNA synthesizer, asolution phase reaction, or combinatorial techniques such as theabove-described SURF technique.

Monomer units bearing protected or unprotected functional groups areprepared as per procedures described in the examples provided below. Ifthe functional group is such that it will react with other moieties orreagents during phosphitylation or oligomerization, the functional groupis appropriately protected with a protecting group. Such protectinggroup is then removed upon completion of the synthesis of oligomericcompound.

To detect an active sequence generated via a combinatorial technique,the concentration of the active molecule is selected to be ofsufficiently great that the molecule can be detected within thesensitivity of the chosen assay. As will be recognized, the number ofunique oligomer sequences within a subset produced via a combinatorialtechnique depends on the length of the oligomer and the number ofdifferent monomers employed. The number of sequences can be determinedby raising the number of monomers to a power equal to the number ofrandom positions. This is illustrated in Table II. Table II alsoindicates the concentration of each sequence when the subsetconcentration is 100 μM, a typical high-test concentration. We havefound that the number of monomers and their length can be based upon anestimate of the expected IC₅₀ (i.e., a concentration at which 50% ofenzyme activity is inhibited) that is desirable in a final oligomericcompound. For an expected IC₅₀ of 100 nM, the complexities shown inTable II are acceptable, that is, the libraries shown in Table II havecomplexities that would allow detection of a unique sequence with anIC₅₀ of about 100 nM or less.

                  TABLE II                                                        ______________________________________                                        Complexity of Libraries                                                                     Sequences                                                                              nM Each Sequence                                       Length        Per Subset                                                                             At 100 μM Subset                                    ______________________________________                                        5 Monomers                                                                    4-mer         125      800                                                    5-mer         625      160                                                    6 Monomers                                                                    4-mer         216      463                                                    5-mer         1,296    77                                                     7 Monomers                                                                    4-mer         343      291                                                    8 Monomers                                                                    4-mer         512      195                                                    10 Monomers                                                                   4-mer         1,000    100                                                    ______________________________________                                    

If five monomers are selected for a library, then the library will havea length of five monomer units, XNNNN, where N is an equal molar mixtureof monomer units and X is a different monomer unit in each of the fivesubsets. For ease in synthesis, the fixed position can be selected asthe right end of the molecule. After assay for inhibition of PLA₂activity as described below, position X is fixed with the residue givingthe greatest inhibition and the next subset is synthesized and screened.The fixed position then shifts towards the left end of the oligomer asunrandomization proceeds. Five rounds of synthesis and screening arerequired to determine a unique inhibitor.

The monomer units of the invention are linked to form oligomericcompounds using standard phosphoramidite chemistry that is used forstandard synthesis of oligonucleotides. Since the coupling rates offunctionalized prolinol monomers may vary, the reactivity of theindividual monomers can adjusted such that equal molar incorporation ofeach monomer at each randomized position is effected. Adjusting for thereactivity of the monomers can be effected as in Examples 107 nd 108. Afurther technique for effecting such adjustment is also disclosed in theU.S. patent application entitled "Random Oligonucleotide Libraries AndMethods of Making The Same," bearing attorney docket number ISIS-1009.The foregoing patent application is being filed concurrently with thisapplication, is commonly assigned, and is incorporated herein byreference.

In a SURF screening strategy the amount of oligomer is selected suchthat the concentration of each subset in the initial round of screeningis relatively high (about 100 μM). It is presently preferred tosynthesize oligomers using a DNA synthesizer. On such synthesizers theoligomers are most conveniently synthesized on a 1 to 4 μmol scale.Given the concentration of a subset of libraries at about 100 μm, theassays preferably are performed in a small volume of less than about 200μL.

Exemplary compounds of the invention are prepared in the followingexamples, which are not intended to be limiting.

EXAMPLE 1 N-Fmoc-trans-4-Hydroxy-L-Proline

trans-4-Hydroxy-L-proline (5.00 g, 38.2 mmol) and NaHCO₃ (8.00 g, 95.2mmol) were suspended in 150 ml H₂ O/Dioxane (1:1). Fluorenylmethylchloroformate (11.4 g, 44.0 mmol) in 25 ml toluene was added dropwise.The temperature of the reaction was not allowed to rise above 25° C.during the addition. The mixture was stirred vigorously overnight, andthen quenched with 50 ml saturated NaHCO₃ solution and 50 ml water. Thesolution was then extracted with 100 ml diethyl ether. The aqueous layerwas acidified to pH 1 with concentrated HCl, and extracted twice withethyl acetate, and the organic extracts washed with brine. The solutionwas dried with MgSO₄, filtered and the solvent removed in vacuo. Thepure product crystallized from the concentrated solution. Yield: 13.4 g(100%). ¹ H NMR: (CDCl₃, 200 MHz) δ 7.75-7.15 (8H, m, ArH), 4.55-4.05(5H, m, ArCHCH₂, H₂, H₄), 3.65-3.45 (2H, m, 2 H5), 2.35-2.10 (2H, m, 2H3).

EXAMPLE 2 N-Fmoc-3-Hydroxypyrrolidine-5-Methanol

To a solution of N-Fmoc-trans-4-hydroxy-L-proline (13.4 g, 38.1 mmol) in250 ml THF was added borane-methyl sulfide (78 mmol, 5.78 g, 7.22 ml)dropwise at room temperature. After the evolution of H₂ had ceased, thesolution was heated to reflux with mechanical stirring. After 1 hour awhite precipitate had formed. Methanol was carefully added, and theresulting solution refluxed for a further 15 minutes. The solution wascooled to room temperature, the solvents evaporated under reducedpressure, and the residual gum coevaporated with 2×100 ml methanol. Theresulting crystalline product weighed 12.0 g (35.3 mmol, 93%). ¹ H NMR:(CDCl3, 200 MHz) δ 7.85-7.25 (8H, m, ArH), 4.50-4.10 (5H, m, ArCHCH2,H3, H5), 3.80-3.40 (4H, m, 2 H2, 2 H6), 2.15-1.95 (1H, m, H2a),1.80-1.60 (1H, m, H2b).

EXAMPLE 3 N-Fmoc-5-Dimethoxytrityloxymethyl-3-Hydroxypyrrolidine

The diol, N-Fmoc-3-hydroxypyrrolidine-5-methanol (10.59 g, 31.2 mmol)was coevaporated with dry pyridine (2×50 ml), redissolved in 200 ml drypyridine, and cooled in an ice bath. Dimethoxytrityl chloride (11.0 g,32.5 mmol) was added in portions over 30 min, and the solution stirredat 0° C. overnight. Methanol was then added (10 ml), and the solventremoved under reduced pressure. The resulting gum was redissolved intoluene (100 ml), filtered to remove the pyridinium hydrochloride andtaken to dryness again. The residue was dissolved in CH₂ Cl₂ (300 ml),washed with 150 ml 0.1M citric acid solution, 150 ml sat NaHCO₃, brine,and dried with MgSO₄ followed by evaporation. The residue wascrystallized from methanol and dried to give (15.4 g, 23.95 mmol, 77%).

EXAMPLE 4 5-Dimethoxytrityloxymethyl-3-Hydroxypyrrolidine

To a solution N-Fmoc-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine(3.40 g, 5.30 mmol) in 15 ml DMF was added piperidine (1.09 ml, 0.935 g,11.0 mmol). The solution was stirred at room temperature for 1 hour,water (100 ml) added, and the aqueous solution extracted with ethylacetate (2×75 ml). The organic extracts were washed with aqueous NaHCO₃,brine, dried with MgSO₄ and evaporated. The residue was purified byflash column chromatography using a gradient of 1→3% MeOH in CH₂ Cl₂containing 0.5% triethylamine. Pure product was obtained (1.86 g, 84%).¹ H NMR: (CDCl₃, 200 MHz) δ 7.42-6.80 (13 H, ArH), 4, 35 (1H, m, H5),3.77 (6H, s, 2 OCH₃), 3.62 (1H, m, H3), 3.13-2.88 (4H, m, 2 H6, 2 H2),1.87 (1H, q, H4a), 1.65 (1H, m, H4b).

EXAMPLE 5 N-Palmitoyl-5-Dimethoxytrityloxymethyl-3-Hydroxypyrrolidine

To the amino alcohol 5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine(0.50 g, 1.19 mmol) dissolved in 5 ml dry pyridine was addedchlorotrimethylsilane (0.227 ml, 194 mg, 1.79 mmol), with stirring for 1hour. The carboxylic acid component (e.g. palmitic acid, 359 mg, 1.40mmol), hydroxybenzotriazole (209 mg, 1.55 mmol) anddimethylaminopropylethylcarbodiimide (EDC) (281 mg, 1.80 mmol) weredissolved in 5 ml DMF (if necessary 5 ml CH₂ Cl₂ co-solvent added) andstirred for 1 hour. This solution was then added to the pyridinesolution of 5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine, and thesolution stirred until complete disappearance of the starting material.The reaction was stopped by addition of 5 ml sat NaHCO₃ and after 15minutes the solution was diluted with water (100 ml), extracted withethyl acetate (2×75 ml), washed with NaHCO₃, brine, dried andevaporated. The product was purified by silica gel chromatography usingethyl acetate/hexane (EtOAc/Hex) as eluant. ¹ H NMR: (CDCl₃, 200 MHz) (2rotamers, 3'-O-TMS) δ 7.43-7.13, 6.88-6.74 (13 Ar--H), 4.67, 4.51, 4.40,4.13 (4 m, 2H, H3, H5), 3.90-3.67 (m, 1H, H2a), 3.80 (s, 6H, OCH₃), 3.45(m, 2H, H2b, H6a), 3.12 (m, 1H, H6b), 2.34-1.78 (m, 4H, H4a, H4b,COCH₂), 1.65, 1.25 (2 s, 26 H, CH₂), 0.87 (t, CH₃), 0.10 (s, 9H, OSi(CH₃) 3).

EXAMPLE 6 N-Isobutyroyl-5-Dimethoxytrityloxymethyl-3-Hydroxypyrrolidine

To a solution of N-hydroxysuccinimide (15 mmol, 1.72 g) in 250 mlpyridine was added isobutyryl chloride (13 mmol, 1.39 g, 1.36 ml). Aprecipitate formed. Diisopropylethylamine (20 mmol, 2.58 g, 3.5 ml) wasadded, the mixture cooled to 0° C., and5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine (5.00 g, 11.9 mmol)added. The solution was stirred until the starting material wasconsumed. The reaction was quenched with sat NaHCO₃, and the solventevaporated. The residue was extracted twice with ethyl acetate, washedwith portions of water, NaHCO₃, 0.1M citric acid, NaHCO₃, brine, driedwith MgSO₄ and evaporated. ¹ H NMR: (CDCl₃, 200 MHz) (2 rotamers,3'-O-TMS) d 7.43-7.13, 6.88-6.74 (13 Ar--H), 4.75, 4.52, 4.37, 4.16 (4m, 2H, H3, H5), 3.78 (s, 6H, OCH₃), 3.88-3.78 (m, 1H, H2a), 3.68-3.34(m, 2H, H2b, H6a), 3.28-2.93 (m, 1H, H6b), 2.67, 2.58 (2 m, 1H,COCH(Me)₂), 2.18-1.80 (2 m, 2H, H4a, b), 1.15, 0.98 (2 q, 6H,COCH(CH₃)₂), 0.10 (d, 9H, OSi(CH₃) ₃).

EXAMPLE 7N-(Phenylacetyl)-5-Dimethoxytrityloxymethyl-3-Hydroxypyrrolidine

Phenylacetic acid (1.50 g, 11 mmol) and HOBT (1.63 g, 12 mmol) weredissolved in 100 ml CH₂ Cl₂ and EDC (15 mmol, 2.88 g) was added. After15 min, 5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine was added,followed by DIEA (20 mmol, 3.5 ml). The reaction was stirred until thestarting material was consumed, and quenched with 10 ml NaHCO₃. Themixture was extracted twice with ethyl acetate, washed with NaHCO₃,brine, dried with MgSO₄, and evaporated. The product was purified byflash chromatography to give 4.0 g product (75%). ¹ H NMR: (CDCl₃, 200MHz) (2 rotamers, 3'-O-TMS) d 7.43-7.13, 6.88-6.74 (13 Ar--H), 4.67,4.49, 4.37, 4.13 (4 m, 2H, H3, H5), 3.78 (s, 6H, OCH₃), 3.78-3.50 (m,2H, H2a, b), 3.66, (s, 2H, CH₂ Ar) 3.35 (q, 1H, H6a), 3.12 (m, 1H, H6b),2.14-1.70 (m, 2H, H4a, b), 0.10 (d, 9H, OSi(CH₃)₃).

EXAMPLE 8 Succinic acid Fluorenylmethyl Ester

Fluorenemethanol (10.0 g, 51.0 mmol) was dissolved in 150 ml CH₂ Cl₂,and succinic anhydride (5.6 g, 56 mmol) was added. The solution wasstirred for 6 h, and a further portion of succinic anhydride (2.5 g, 25mmol) was added, and stirring continued overnight. The reaction appearedcomplete by TLC. The solvent was then removed, and the residue extractedwith ethyl acetate, washed with 1% HCl, water, brine, dried (MgSO₄) andevaporated to an oil which crystallized on standing. A quantitativeyield of crude product was obtained which was used without furtherpurification.

EXAMPLE 9N-(Fluorenylmethylsuccinoyl)-5-Dimethoxytrityloxymethyl-3-Hydroxypyrrolidin

Succinic acid fluorenylmethyl ester (4.27 g, 14.4 mmol) and HOBT (2.14g, 15.8 mmol) were dissolved in 100 ml CH₂ Cl₂. EDC was added (3.03 g,15.8 mmol), and the solution stirred for 30 min. DIEA (2.79 ml, 2.07 g,16 mmol) was added, followed by5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine (5.00 g, 12 mmol). After30 min, sat NaHCO₃ was added (10 ml) and the solvents removed 10 minuteslater. The residue was extracted with ethyl acetate, washed with NaHCO₃,brine, dried with MgSO₄, and evaporated. The product was purified byflash chromatography (2% MeOH in CH₂ Cl₂) to give 6.1 g product (8.7mmol, 73%). ¹ H NMR: (CDCl₃, 200 MHz) (2 rotamers) d 7.80-6.80 (21Ar--H), 4.62, 4.46, 4.21 (3 m, 2H, H3, H5), 4.41-4.28 (m, 3H, CH₂ CH),3.78 (s, 6H, OCH₃), 3.92, 3.71, 3.54, 3.41, 3.16 (5 m, 4H, H2a,b,H6a,b), 2.88-2.75 (m, 4H, COCH₂ H₂ CO), 2.50-1.93 (4m, 2H, H4a, b).

EXAMPLE 10 (N-1-Thymine)-2-Acetic Acid

Methyl bromoacetate (25.5 g, 15.2 ml, 160 mmol) was added to asuspension of K₂ CO₃ (44.2 g, 320 mmol) and thymine (20.2 g, 160 mmol)in 500 ml dry DMF with stirring overnight. The suspension was filteredand the solvent removed under reduced pressure. The residue wassuspended in 120 ml H₂ O and 30 ml 4N HCl, stirred for 30 minutes andfiltered again. The solid was suspended in 250 ml H₂ O, to which wasadded 100 ml 2.5M NaOH. The solution was heated to boiling, cooled andacidified to pH 1 with concentrated HCl. The precipitate was dried invacuo to give 13.6 g (73.6 mmol, 46%) pure product. ¹ H NMR: (DMSO-d6,200 MHz) δ 7.48 (s, 1H, H6), 4.37 (s, 2H, CH₂), 1.76 (s, 3H, CH₃).

EXAMPLE 11 N-(N1-Thymine)-2-acetyl!-5-Dimethoxytrityloxymethyl-3-Hydroxypyrrolidine

Thymine acetic acid (1.36 g, 7.4 mmol) and HOBT (1.08g, 8.0 mmol) weredissolved in 25 ml DMF, and EDC (1.72 g, 9.0 mmol) was added. A thickwhite precipitate formed, and stirring was continued for 1 h. In aseparate flask, 5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine (2.59 g,6.17 mmol) and TMS-Cl (9.0 mmol, 0.98 g, 1.14 ml) were stirred for onehour in 20 ml pyridine, and added to the DMF solution. The combinedsolution was stirred overnight at RT, and quenched with 10 ml NaHCO₃.After 1 hr, the solution was diluted with water, and extracted twicewith ethyl acetate, washed with NaHCO₃, 0.1M citric acid, NaHCO₃, brine,dried with MgSO₄ and evaporated. The product was isolated by flashchromatography (3% MeOH/CH₂ Cl₂) to give 3.07 g (85%). ¹ H NMR:(DMSO-d6, 200 MHz) (2 rotamers) d 7.50-6.80 (C6H, 13 Ar--H), 4.60-4.40,4.28 (m, 3H, COCH₂, H5), 4.15 (m, 1H, H3), 3.70 (s, 6H, OCH₃), 3.66 (m,1H, H2a), 3.45-2.94(m, 4H, H2a, b, H6a, b), 2.08-1.82 (m, 2H, H4a, H4b),1.65 (s, 3H, (C5)CH₃).

EXAMPLE 12 N-Fmoc-3-Aminopropionic Acid

Sodium bicarbonate (2.52 g, 30 mmol) and 3-aminopropionic acid (1.00 g,11.2 mmol) were dissolved in 50 ml water and 50 ml dioxane was added. Asolution of fluorenylmethyl chloroformate (3.10 g, 12.0 mmol) in 50 mldioxane was added dropwise with stirring. After 6 hours the solution wasdiluted with water (100 ml) and saturated bicarbonate solution (50 ml),extracted once with diethyl ether, and the aqueous layer acidified to pH2 with concentrated HCl. The cloudy solution was extracted with ethylacetate (2×100 ml), washed with brine and dried with MgSO₄. Afterevaporation a mixture of the title product and the peptide dimer wasobtained. The pure product was obtained by flash chromatography. ¹ HNMR: (CDCl₃, 200 MHz) δ 7.95-7.26 (8H, m, ArH), 7.40-7.15 (3H, m, CHCH₂O), 3.20 (2H, t, J=8 Hz, C₂ N), 2.40 (2H, t, J=8 Hz, HOOCCH₂).

EXAMPLE 13N-Fmoc-3-Aminopropionoyl-5-Dimethoxytrityloxymethyl-3-Hydroxypyrrolidine

This compound was prepared as per the procedure described in Example 5using N-Fmoc-3-aminopropionic acid as the carboxylic acid component. ¹ HNMR: (CDCl₃, 200 MHz) (2 rotamers) δ 7.80-6.80 (21 Ar--H), 5.40 (br s,1H, CONH), 4.62, 4.51 (2 m, 1H, H5), 4.41-4.28 (m, 3H, CH₂ CH), 4.20 (m,1H, H3), 3.78 (s, 6H, OCH₃), 3.92, 3.65 (2 m, 2H, H2a), 3.70-3.30 (m,4H, H2b, COCH₂ CH₂ NHCO, H6a), 3.15 (m, 1H, H6b), 2.60-1.90 (3m, 4H,H4a, b, COCH₂ CH₂ NHCO).

EXAMPLE 14 N-Imidazolyl 2-Acetic acid

Imidazole (3.7 g, 54 mmol) was added to a suspension of sodium hydride(2.6 g of a 60% dispersion in oil, 60 mmol) in 50 ml dry THF.Bromoacetic acid (3.4 g, 24 mmol) was then added and the mixture stirredovernight. Water (1 ml) was then added and the solvent removed underreduced pressure. The residue was taken up in water (50 ml, pH>10),extracted with ether and the organic layer discarded. The aqueous layerwas acidified to pH 1 with concentrated HCl and extracted again withether. The aqueous layer was evaporated to dryness. The oily residue wasdissolved in absolute ethanol (EtOH) to precipitate NaCl, andrecrystallized from acetone/methanol to give 1.22 g (7.5 mmol, 30%) pureproduct as the hydrochloride. ¹ H NMR: (DMSO-d6, 200 MHz) δ 9.20 (s,H2), 7.76 (d, J=1.5 Hz), 7.69 (d, J=1.5 Hz), 5.20 (s, CH₂).0

EXAMPLE 15N-(1-Imidazolyl-2-Acetyl)-5-Dimethoxytrityloxymethyl-3-Hydroxypyrrolidine

Imidazoleacetic acid (1.00 g, 6.15 mmol) and HOBT (1.01 g, 7.5 mmol)were dissolved in 25 ml DMF, and EDC (1.76 g, 9.2 mmol) was added. Athick white precipitate formed, and stirring was continued for 1 h. In aseparate flask, 5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine (2.52 g,6.0 mmol) and TMS-Cl (7.5 mmol, 0.82 g, 0.95 ml) were stirred for onehour in 20 ml Pyridine, and added to the DMF solution. The combinedsolution was stirred overnight at RT, and quenched with 10 ml NaHCO₃.After 1 hr, the solution was diluted with water, and extracted twicewith ethyl acetate, washed with NaHCO₃, 0.1M citric acid, NaHCO, brine,dried with MgSO₄ and evaporated. The product was isolated by flashchromatography (5% MeOH/CH₂ Cl₂) to give 2.07 g (65%). ¹ H NMR:(DMSO-d6, 200 MHz) (2 rotamers) d 7.50-6, 80 (3 Imidazole-H, 13 Ar--H),4.90 (ABq, 2H, COCH₂), 4.44 (m, 1H, H5), 4.28 , 4.15 (m, 1H, H3), 3.70(s, 6H, OCH₃), 3.66 (m, 1H, H2a), 3.39 (m, 1H, H2b), 3.35-3.00 (m, 2H,H6a, H6b), 2.08-1.82 (m, 2H, H4a, H4b).

EXAMPLE 16 (9-Adenine)-2-Acetic Acid Ethyl Ester

Sodium hydride (8.20 g 60% in oil, 205 mmol) was added to a suspensionof adenine (25.0 g, 185 mmol) in 500 ml DMF. After vigorous stirring for2 hours using a mechanical stirrer, H₂ evolution stopped and a thickslurry was obtained. Ethyl bromoacetate (55.6 g, 36.9 ml, 333 mmol) wasadded dropwise over 3 hours, and stirring continued for a further 1hour. Water (10 ml) and H₂ SO₄ were added to pH 4. The solvent wasevaporated and the residue suspended in 500 ml H₂ O, filtered and washedwith water. The residue was recrystallized from 400 ml ethanol to give23.8 g (108 mmol, 58%) pure product.

EXAMPLE 17 (N6-Benzoyl-9-Adenine)-2-Acetic Acid

To a suspension of (9-adenylyl)-2-acetic acid ethyl ester (6.06 g, 27.4mmol) in 250 ml dry pyridine was added benzoyl chloride (9.60 ml, 11.6g, 82 mmol), and the solution stirred for 4 hours at room temperature.Methanol (25 ml) was added and the solvents evaporated. The residue wasdissolved in ethyl acetate (2×250 ml), washed with 0.1N HCl, H₂ O,saturated NaHCO₃, brine, and dried with Na₂ SO₄. The organic extractswere evaporated and the solid residue was redissolved in 250 ml THF at0° C., to which was added 100 ml 1M NaOH. The solution was stirred at 0°C. for 1 hour and acidified to pH 1 with concentrated HCl, and theaqueous portion extracted once with ether. The product, which began tocrystallize almost immediately, was collected by filtration to yield4.96 g (61%). ¹ H NMR: (DMSO-d6, 200 MHz) δ 8.86, 8.84 (d, H2, H8), 8.1(d, 2H, J=7.0 Hz, ArH), 7.69-7.58 (m, 3H, Ar--H), 5.22 (s, 2H, CH₂).

EXAMPLE 18 N- (N-6-Benzoyl9-Adeninyl)-2-Acetyl!-5-Dimethoxytrityloxymethyl-3-Hydroxypyrrolidine

(N-6-Benzoyl-9-Adeninyl)-2-acetic acid (2.76 g, 9.30 mmol and HOBT (1.49g, 11.0 mmol) were dissolved in 50 ml DMF, and DCC (2.89 g, 14.0 mmol)was added. A thick white precipitate formed, and stirring was continuedfor 1 h. In a separate flask,5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine (3.00 g, 7.15 mmol) andTMS-Cl (8.6 mmol, 0.93 g, 1.09 ml) were stirred for one hour in 50 mlPyridine, and added to the DMF solution. The combined solution wasstirred overnight at RT, and quenched with 25 ml NaHCO₃. After 1 hr, thesolution was diluted with water, and extracted twice with ethyl acetate,washed with NaHCO₃, 0.1M citric acid, NaHCO₃, brine, dried with MgSO₄and evaporated. The product was isolated by flash chromatography (2%MeOH/CH₂ Cl₂) to give 2.03 g (42%). ¹ H NMR: (DMSO-d6, 200 MHz) (2rotamers) d 8.67, 8.34 (2 s, 2H, Adenine H2, H8) 8.04 (d, 2H, BenzoylH2, H6), 7.54 (m, 3H, Benzoyl H3, 4, 5), 7.35-7.10, 6.84 (13 Ar--H),5.22 (ABq, 2H, COCH₂), 4.50, 4.34 (2 m, 1H, H5), 4.23 , 4.15 (2 m, 1H,H3), 4.05, 3.80 (m, 1H, H2a), 3.70 (s, 6H, OCH₃), 3.93, 3.55 (m, 1H,H2b), 3.35, 3.18 (m, 2H, H6a), 3.28, 2.98 (m, 1H, H6B), 2.30-1.84 (4 m,2H, H4a, H4b).

EXAMPLE 19 N-4-Benzoylcytosine

Cytosine hemihydrate (12.0 g, 100 mmol) was coevaporated with pyridineand resuspended in 250 ml dry pyridine. Benzoyl chloride (58 ml, 70.3 g,500 mmol) was added dropwise (exothermic). The solution was stirred atRT overnight, and water (50 ml) carefully added. The solvent wasevaporated, and the residue dissolved in 700 ml H₂ O containing 55 gNaOH. The solution was stirred for 1 h after complete dissolution of thematerial. Concentrated HCl was then added to pH 4.0, the whiteprecipitate was collected and boiled in 1 liter EtOH, cooled to RT andfiltered to give 16.1 g product (75%).

EXAMPLE 20 N-4-Benzoyl-1-Cytosine-2-Acetic acid

To a suspension of N-4-Benzoylcytosine (15.0 g, 69.7 mmol) and K₂ CO₃(9.7 g, 70 mmol) in 500 ml DMF was added methyl bromoacetate (6.6 ml,10.7 g, 70 mmol). The suspension was stirred vigorously for 3 days,filtered and evaporated. The residue was treated with water (120 ml),and 10 ml 4N HCl for 15 min, and the solid collected by filtration. Theresidue was resuspended in 120 ml water, and 60 ml 2N NaOH added. Thesuspension was stirred at RT for 45 min, until all the solids haddissolved. The solution was acidified to pH 2 with conc HCl, filtered,and the solid dried in vacuo at 60° C. to give 11.6 g product (61%).

EXAMPLE 21 N-(N-4-Benzoyl-1-Cytosine)-2-Acetyl!-5-Dimethoxytrityloxymethyl-3-Hydroxypyrrolidine

(N-4-Benzoyl-1-Cytosine)-2-acetic acid (3.85 g, 14.0 mmol) and HOBT(2.25 g, 16.6 mmol) were dissolved in 50 ml DMF, and EDC (3.83 g, 20.0mmol) was added. A thick white precipitate formed, and stirring wascontinued for 1 h. In a separate flask,5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine (5.00 g, 11.9 mmol) andTMS-Cl (13 mmol, 1.41 g, 1.65 ml) were stirred for one hour in 50 mlPyridine, and added to the DMF solution. The combined solution wasstirred overnight at RT, and quenched with 25 ml NaHCO₃. After 1 hr, thesolution was diluted with water, and extracted twice with ethyl acetate,washed with NaHCO₃, 0.1M citric acid, NaHCO₃, brine, dried with MgSO₄and evaporated. The product was isolated by flash chromatography (2%MeOH/CH₂ Cl₂) to give 6.10 g product (75%).

EXAMPLE 22 N-2-Isobutyroyl-9-Guanine-2-Acetic Acid

To a suspension of 2-amino-6-chloropurine (10 mmol) and K₂ CO₃ (15 mmol)in DMF (25 ml) is added ethyl bromoacetate (10 mmol). The mixture isstirred vigorously for 24 hours, filtered and the solvent evaporated.The residue is resuspended in 25 ml pyridine and isobutyroyl chlorideadded (20 mmol). After stirring for 18 hours, water is added and thesolvent removed. The residue is suspended in 1N HCl and heated to refluxfor 1 hour. The suspension is then cooled to 0° C., NaOH added to pH 12,and the suspension stirred for 1 hour. The solution is acidified to pH3, and the product is collected by filtration.

EXAMPLE 23 N-(N2-Isobutyroyl-9-Guanine)-2-Acetyl5-Dimethoxytrityloxymethyl-3-Hydroxypyrrolidine

The title compound is prepared as per the procedure of Example 5 usingN2-isobutyroyl-9-guanine-2-acetic acid as the carboxylic acid component.

EXAMPLE 24 Benzyl 3, 6, 9, 12-Tetraoxatridecanoate

Triethyleneglycol monomethyl ether (10 mmol) and benzyl bromoacetate (11mmol) are added to a suspension of anhydrous K₂ CO₃ (15 mmol) in 50 mlanhydrous DMF. The suspension is stirred at room temperature overnight.Water is added and the emulsion is extracted with ethyl acetate (3×200ml), washed with water, brine, and dried with MgSO₄. The solvent isevaporated and the residual oil purified by flash chromatography to givethe title compound.

EXAMPLE 25 3, 6, 9, 12-Tetraoxatridecanoic Acid

Benzyl-3, 6, 9, 12-Tetraoxatridecanoate (5 mmol) is dissolved inmethanol (50 ml) and 10% palladium on carbon is added (100 mgcatalyst/mmol). The suspension is shaken under 30 psi H₂ until thestarting material is consumed. The suspension is filtered through ashort pad of Celite, washed thoroughly with methanol, and the solventevaporated. The product is used directly without purification.

EXAMPLE 26 N- 3, 6, 9,12-Tetraoxatridecanoyl!-5-Dimethoxytrityloxymethyl-3-Hydroxypyrrolidine

The title compound is prepared via the procedure of Example 5 using 3,6, 9, 12-tetraoxatridecanoic acid as the carboxylic acid component.

EXAMPLE 27 Benzyl Bis- (2-Pyridyl)-2-ethyl!-Aminoacetate

To a suspension of K₂ CO₃ (15 mmol) in 25 ml DMF was added2,2'-bis(2-pyridylethyl)-amine (10 mmol) followed by benzyl bromoacetate(12 mmol). The suspension was stirred for 4 hours at room temperature.Water was then added, and the suspension extracted with ethyl acetate(2×100 ml), washed with 5% Na₂ CO₃, water, brine, dried with MgSO₄ andthe solvents removed. The product was obtained as an oil in quantitativeyield. Product was identified by NMR.

EXAMPLE 28 Bis(2-(2-Pyridyl)ethyl)-Aminoacetic Acid

Benzyl bis- (2-pyridyl)-2-ethyl!-aminoacetate (5 mmol) is dissolved inmethanol (50 ml) and 10% palladium on carbon is added (100 mgcatalyst/mmol). The suspension is shaken under 30 psi H₂ until thestarting material is consumed. The suspension is filtered through ashort pad of Celite, washed thoroughly with methanol, and the solventevaporated. The product is used directly without purification.

EXAMPLE 29 N-Bis(2-(2-Pyridyl)ethyl)-Aminoacetyl!-5-Dimethoxytrityloxymethyl-3-Hydroxypyrrolidine

This compound is prepared via the procedure of Example 5 usingbis(2-(2-pyridyl)ethyl)-aminoacetic acid as the carboxylic acidcomponent.

EXAMPLE 30N-(Toluenesulfonyl)-5-Dimethoxytrityloxymethyl-3-Hydroxypyrrolidine

Chlorotrimethylsilane (6.0 mmol) is added dropwise to a solution of5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine (5.0 mmol) in 25 ml drypyridine. After stirring one hour, toluenesulfonyl chloride (6.0 mmol)is added in portions, and stirring continued for two hours. The reactionis quenched with saturated aqueous NaHCO₃, and the mixture stirred untilthe silyl ethers were hydrolyzed. The solvent is removed in vacuo, andthe residue partitioned between water and ethyl acetate. The organiclayer is washed with NaHCO₃, water, brine and dried with Na₂ SO₄. Thesolvent is removed and the resulting oil purified by flashchromatography, using a gradient of MeOH in CHCl₃.

EXAMPLE 31N-(Trifluoromethanesulfonyl)-5-Dimethoxytrityloxymethyl-3-Hydroxypyrrolidine

Chlorotrimethylsilane (6.0 mmol) is added dropwise to a solution of5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine (5.0 mmol) andtriethylamine (15 mmol) in 50 ml dry CH₂ Cl₂. After 1 hour the solutionis cooled to -78° C., and trifluoromethanesulfonic anhydride (5.5 mmol)is added dropwise. The cooling bath is removed and the mixture allowedto warm to room temperature. The crude product is dissolved in pyridineand NaHCO₃ solution is added to hydrolyze the TMS ether. The solvent isevaporated, the residue partitioned between ethyl acetate and water,washed with NaHCO₃, brine and dried with MgSO₄. The residue is purifiedby flash chromatography using a gradient of methanol in CHCl₃.

EXAMPLE 32 N-Benzyl-5-Dimethoxytrityloxymethyl-3-Hydroxypyrrolidine

Chlorotrimethylsilane (6.0 mmol) is added dropwise to a solution of5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine (5.0 mmol), imidazole (5mmol) and triethylamine (15 mmol) in 25 ml dry DMF. After stirring onehour the solvent is removed in vacuo, and the residue redissolved inacetonitrile (25 ml) and triethylamine (10 mmol). Benzyl bromide (6.0mmol) is added, and stirring continued overnight. The reaction isquenched with saturated aqueous NaHCO₃, and the mixture stirred untilthe silyl ethers were hydrolyzed. The solvent is removed in vacuo, andthe residue partitioned between water and ethyl acetate. The organiclayer is washed with NaHCO₃, water, brine and dried with Na₂ SO₄. Thesolvent is removed and the resulting oil purified by flashchromatography, using a gradient of MeOH in CHCl₃.

EXAMPLE 33N-(Aminocarbonyl)-5-Dimethoxytrityloxymethyl-3-Hydroxypyrrolidine

Chlorotrimethylsilane (6.0 mmol) is added dropwise to a solution of5-DMT-hydroxypyrrolidine (5.0 mmol) and triethylamine (15 mmol) in 50 mldry CH₂ Cl₂. After one hour, dimethylaminopyridine (1 mmol) is addedfollowed by trimethylsilyl isocyanate (5.5 mmol). The solution isstirred until the starting material is consumed. The solvent is removedin vacuo and the crude product redissolved in pyridine and NaHCO₃solution to hydrolyze the TMS ethers. The solvent is evaporated, theresidue partitioned between ethyl acetate and water, washed with NaHCO₃,brine and dried with MgSO₄. The residue is purified by flashchromatography using a gradient of methanol in CHCl₃.

EXAMPLE 34N-(Methylaminothiocarbonyl)-5-Dimethoxytrityloxymethyl-3-Hydroxypyrrolidine

Chlorotrimethylsilane (6.0 mmol) is added dropwise to a solution of5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine (5.0 mmol) andtriethylamine (15 mmol) in 50 ml dry CH₂ Cl₂. After one hour,dimethylaminopyridine (1 mmol) is added followed by methylisothiocyanate(5.5 mmol). The solution is stirred until the starting material isconsumed. The solvent is removed in vacuo and the crude productredissolved in pyridine and NaHCO₃ solution to hydrolyze the TMS ethers.The solvent is evaporated, the residue partitioned between ethyl acetateand water, washed with NaHCO₃, brine and dried with MgSO₄. The residueis purified by flash chromatography using a gradient of methanol inCHCl₃.

EXAMPLE 35 N-(Benzyloxycarbonyl)-3-Hydroxypyrrolidine-5-Methanol

To a solution of N-CBz-4-hydroxy-L-proline (38.1 mmol) in 250 ml THF wasadded borane-methyl sulfide (78 mmol) dropwise at room temperature.After the evolution of H₂ had ceased, the solution was heated to refluxwith mechanical stirring. After 1 hour a white precipitate had formed.Methanol was carefully added, and the resulting solution refluxed for afurther 15 minutes. The solution was cooled to room temperature, thesolvents evaporated under reduced pressure, and the residual gumcoevaporated with 2×100 ml MeOH. The product was obtained as a viscousoil in quantitative yield and identified by NMR.

EXAMPLE 36N-(Benzyloxycarbonyl)-5-Dimethoxytrityloxymethyl-3-Hydroxypyrrolidine

N-(Benzyloxycarbonyl)-3-hydroxypyrrolidine-5-methanol (35.3 mmol) wascoevaporated with dry pyridine (2×50 ml), redissolved in 200 ml drypyridine, and cooled in an ice bath. Dimethoxytrityl chloride (38 mmol)was added in portions over 15 minutes, and the solution stirred at roomtemperature overnight. Methanol was then added (10 ml), and the solventremoved under reduced pressure. The resulting gum was redissolved intoluene (100 ml), filtered to remove the pyridinium hydrochloride andtaken to dryness again. The residue was chromatographed (0 to 1.5%MeOH/CH₂ Cl₂) to give the product. The product was identified by NMR.

EXAMPLE 37 N-α-(FMOC)-glutamic acid γ-benzyl ester

To a solution of γ-benzyl glutamate (10 mmol) in 50 ml dioxane and 50 mlwater is added triethylamine (25 mmol), followed by a solution offluorenylmethyl chloroformate (11 mmol) in 50 ml dioxane. The mixture isvigorously stirred until the starting material is consumed. The solutionis acidified to pH 2 with concentrated HCl, extracted with ethyl acetate(2×250 ml), washed with brine, dried with MgSO₄ and evaporated. Theproduct is used without purification.

EXAMPLE 38 N-α-(FMOC)-γ-benzyl-L-glutamic acid fluorenylmethyl ester

N-α-(FMOC)-glutamic acid δ-benzyl ester (5 mmol), fluorenylmethanol (5.5mmol) and dimethylaminopyridine (0.5 mmol) are dissolved in 50 ml CH₃Cl₂. Dimethylaminopropyl ethyl carbodiimide (EDC, 6.0 mmol) is added,and the solution stirred at room temperature. After complete consumptionof the starting material the solution is diluted with CH₂ Cl₂, washedwith 1% HCl, water and brine, dried with MgSO4 and evaporated. Theresidue is purified by flash chromatography using ethyl acetate andhexane as eluant.

EXAMPLE 39 N-α-(FMOC)-L-glutamic acid α-fluorenylmethyl ester

N-α-(FMOC)-γ-benzyl-L-glutamic acid fluorenylmethyl ester (5 mmol) isdissolved in methanol (50 ml) and 10% Palladium on carbon is added (100mg catalyst/mmol). The suspension is shaken under 30 psi H₂ until thestarting material is consumed. The suspension is filtered through ashort pad of Celite, washed thoroughly with methanol, and the solventevaporated. The product is used directly without purification.

EXAMPLE 40N-(N-α-Fmoc-α-Fluorenylmethyl-γ-glutamyl)-5-Dimethoxytrityloxymethyl-3-Hydroxypyrrolidine

The title compound is prepared by the procedure of Example 5 usingN-α-(FMOC)-L-glutamic acid α-fluorenylmethyl ester as the carboxylicacid component.

EXAMPLE 41 N-Carbazolyl-2-Acetic acid

The title compound is prepared as per Example 14 using carbazole as thestarting heterocycle.

EXAMPLE 42N-(N-Carbazolyl-2-Acetyl)-5-Dimethoxytrityloxymethyl-3-Hydroxypyrrolidine

This compound is prepared as per the procedure described in Example 5using N-carbazolyl-2-acetic acid as the carboxylic acid component.

EXAMPLE 43 N-Pyrrolyl-2-Acetic acid

The title compound is prepared as per Example 14 using pyrrole as thestarting heterocycle.

EXAMPLE 44N-(N-Pyrrolyl-2-Acetyl)-5-Dimethoxytrityloxymethyl-3-Hydroxypyrrolidine

This compound is prepared as per the procedure described in Example 5using N-pyrrolyl-2-acetic acid as the carboxylic acid component.

EXAMPLE 45N-(2-Naphthylacetyl)-5-Dimethoxytrityloxymethyl-3-Hydroxypyrrolidine

2-Naphthylacetic acid (3.35 g, 18 mmol) and HOBT (2.70 g, 20 mmol, driedunder vacuum at 50° C. 24 h) were suspended in 100 ml CH₂ Cl₂. EDC (3.83g, 20 mmol) was added followed by diisopropylethylamine (3.4 ml, 2.5 g,20 mmol), and the suspension stirred at RT. The DMT-hydroxyprolinol(5.00 g, 12 mmol) was then added after 15 min. After 1 h stirring thereaction was complete by TLC, and the reaction quenched with sat NaHCO₃(10 ml). The solvent was removed after 1 h, the residue was extractedwith ethyl acetate, washed with sat NaHCO₃, water and brine. The organiclayer was dried with MgSO₄, evaporated, and purified by flashchromatography (EA/Hex 3:1) to give pure product (6.7 g, 98%). ¹ H NMR:(DMSO-d6, 200 MHz) (2 rotamers) d 7.8-6.75 (20 H, Ar--H), 4.40 (1H, m,H3), 4.32, 4.20 (1H, m, H5), 3.80 (2H, q, COCH₂), 3.74 (6H, s, OCH₃),3.50 (1H, m, H2a), 3.35 (2H, m, H2b, H6a), 3.05 (1H, m, H6b), 2.00, (1H,m, H4a), 1.83 (1H, m, H4b).

EXAMPLE 46N-(Methanesulfonyl)-5-Dimethoxytrityloxymethyl-3-Hydroxypyrrolidine

5-Dimethoxytrityloxymethyl-3-hydroxypyrrolidine (5.00 g, 12 mmol) andpyridine (1.46 ml, 1.42 g, 18 mmol) were dissolved in 150 ml CH₂ Cl₂ andcooled to 0° C. Freshly distilled methanesulfonyl chloride (0.97 ml,1.43 g, 12.5 mmol) was added dropwise, and the solution allowed to warmto RT. The reaction was quenched with water, evaporated, and extractedwith ethyl acetate, washed with NaHCO₃, water and brine, dried (MgSO₄)and evaporated. The product was purified by flash chromatography(EA/Hex, 2:1) to give 4.33 g product (73%). ¹ H NMR: (CDCl₃, 200 MHz) d7.38-6.80 (13 H, ArH), 4.48 (1H, m, H3), 3.96 (1H, m, H5), 3.70 (8H, s,2 OCH₃), 3.56 (1H, dd, H6a), 3.48 (2H, dd, H6b ), 3.30 (2H, m, 2 H6),2.86 (s, 3H, CH₃), 2.13 (2H, m, 2 H4).

EXAMPLE 47 N-Trifluoroacetyl-Glycine Triethylammonium salt

To a suspension of glycine (1.50 g, 20 mmol) in 100 ml dry methanol wereadded triethylamine (3.5 ml, 2.5 g, 25 mmol) and ethyl trifluoroacetate(3.0 ml, 3.55 g, 25 mmol). The mixture was stirred overnight to give ahomogeneous solution. The solvents were removed and the resulting oilcoevaporated with toluene to remove traces of methanol. The product wasused without purification.

EXAMPLE 48N-(N'-Trifluoroacetylglycyl)-5-Dimethoxytrityloxymethyl-3-Hydroxypyrrolidine

Trifluoroacetylglycine triethylammonium salt N-trifluoroacetyl-glycinetriethylammonium salt (20 mmol) was dissolved in 100 ml CH₂ Cl₂.N-Hydroxysuccinimide (2.30 g, 20 mmol) and DMAP (0.25 g, 2 mmol) wereadded, followed by EDC (4.2 g. 22 mmol). After 30 min stirring at RT,DIEA was added (4.35 ml, 3.25 g, 25 mmol) followed by5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine (5.0 g, 12 mmol). After30 min the starting material was consumed, and the reaction was quenchedwith 10 ml sat NaHCO₃. After 15 min the solvents were removed, and theresidue extracted with ethyl acetate, washed with NaHCO₃, 0.1M citricacid, NaHCO₃, water, brine and dried with MgSO4. Pure product wasobtained in 87% yield (5.95 g, 10.4 mmol). ¹ H NMR: (DMSO-d6, 200 MHz)(2 rotamers) d 7.40-6.80 (13 Ar--H), 4.39, 4.25 (2 m, 1H, H3), 4.12 (m,1H, H5), 3.95 (q, 2H, COCH₂ N), 3.70 (s, 6H, OCH₃), 3.60 (m, 1H, H2a),3.35, 3.27 (2m, 1H, H2b), 3.12 (m, 1H, H6a), 2.98 (m, 1H, H6b), 1.95,1.85 (2m, 2H, H4).

EXAMPLE 49 2-Dimethoxytrityl ethanol

Anhydrous ethylene glycol (2.45 ml, 44 mmol) was dissolved in 250 mlPyridine and cooled to 0° C. TEA (7 ml, 50 mmol) and DMAP (250 mg) wereadded, followed by DMT-Cl (7.4 g, 22 mmol) in portions over 30 min. Thesolution was stirred at 0° C. for 2 h, 10 ml water added and the solventevaporated. The residue was extracted with EtOAc, washed with NaHCO3,water, brine, dried with MgSO4 and evaporated. The residue wascoevaporated with toluene. The product was purified by flashchromatography to give 5.5 g product (70%). ¹ H NMR (CDCl₃); d7.50-7.20, 6.90-6.80 (m, 13 H, ArH), 3.80 (s, 6 H, OCH₃), 3.75 (t, 2H,CH₂ OH), 3.25 (t, 2H, DMTOCH₂).

EXAMPLE 50 2-Dimethoxytrityl ethanol hemisuccinate Triethylammonium salt

A solution of 2-O-(dimethoxytrityl) ethanol (1.0 g, 2.77 mmol),triethylamine (0.4 ml, 3 mmol), and 4-dimethylaminopyridine catalyst(120 mg, 1 mmol) in dry dichloroethane was treated with succinicanhydride (410 mg, 0.41 mmol). The mixture was stirred at 50° C. for 1.5hr and then cooled to room temperature. The mixture was kept at roomtemperature for 16 hrs. The mixture is filtered and the filtrate waspurified by silica gel flash column chromatography usingchloroform-methanol-triethylamine to yield the title compound as atriethylammonium salt. ¹ H NMR: (CDCl₃) δ 7.50-7.20, 6.90-6.80 (m, 13 H,ArH), 4.26 (t, 2H, CH₂ OCO), 3.80 (s, 6 H, OCH₃), 3.25 (t, 2H, DMTOCH₂),3.05 (q, 6H, N(CH₃ CH₃)₃), 2.70 (m, 4H, OOCCH₂ CH₂ COO), 1.25 (t, 9H,N(CH₂ CH₃)₃.

EXAMPLE 51 2-Dimethoxytrityl ethanol H-Phosphonate

A solution of imidazole (4.29 g, 63 mmol)in dry acetonitrile at 0° C.(100ml) was treated dropwise with PCl₃ (1.77 ml, 20.3 mmol) over aperiod of 30 minutes. The resulting solution is further treated withtriethylamine (9.06 ml, 65 mmol). To the thick slurry was added2-O-(dimethoxytrityl)ethanol (2.10 g, 5.81 mmol) in anhydrousacetonitrile (150 ml) slowly over a period of 30 minutes. The mixture isallowed to warm to room temperature and stirred for 15 minutes. Themixture is quenched with 1M TEAB and the mixture is evaporated in vacuoto a minimum volume and extracted with dichloromethane (2×150 ml). Thedichloromethane extracts are washed with TEAB and evaporated in vacuo.The residue was purified by flash column chromatography using a gradientof 0% to 5% methanol in dichloromethane/1% triethylamine to yield 1.3 gpurified material (43%).

¹ H NMR (CDCl₃); d 7.50-7.20, 6.90-6.80 (m, 13 H, ArH), 6.96 (d, 1H,J_(PH) =624 Hz, PH), 4.06 (m, 2H, CH₂ OP), 3.80 (s, 6 H, OCH₃), 3.25 (t,2H, DMTOCH₂), 3.05 (q, 6H, N(CH₂ CH₃)₃), 1.25 (t, 9H, N(CH₂ CH₃)₃). ³¹ pNMR (CDCl₃); 5.89.

EXAMPLE 52N-(1-Methylpyrrole-2-Carbonyl)-5-Dimethoxytrityloxymethyl-3-Hydroxypyrrolidine

To a stirred solution of 1-Methylpyrrole-2-carboxylic acid (1.88 g, 15.0mmol) and HOBT (3.45 g, 18.0 mmol) in 75 ml CH₂ Cl₂ was added EDC (2.43g, 18.0 mmol) and DIEA (3.2 ml, 2.3 g, 18 mmol). After 30 min,5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine (5.00 g, 11.9 mmol) wasadded in portions over 5 min, and stirred for 1 h. The reaction wasquenched after 1 h with 10 ml NaHCO₃, evaporated and the residueextracted twice with EtOAc, washed with NaHCO₃, brine, dried with MgSO4and evaporated. The product was purified by flash chromatography usingEtOAc/Hexane (4:1) as eluant to give 6.2 g product (99%). ¹ H NMR: (DMSOd6, 200 MHz) d 7.38-6.80 (13 H, ArH), 6.95, 6.44, 6.03 (3H, pyrrole-H),4.48 (1H, m, H3), 4.28 (1H, m, H5), 3.77 (11H, s, 2 OCH₃, 2 H2, N--CH₃),3.20 (1H, m, H6a), 2.95 (2H, m, H6b), 2.05 (1H, m, H4a), 1.95 (1H, m,H4b).

EXAMPLE 53 3, 6, 9-Trioxadecyl-4-Toluenesulfonate

Toluenesulfonyl chloride (7.15 g, 37.5 mmol) was added to a solution oftriethyleneglycol monomethyl ether (5.0 ml, 5.13 g, 31.2 mmol) in 100 mlpyridine. The solution was stirred at RT overnight, the solvent removedand the residue extracted twice with CH₂ Cl₂, washed with 0.1N HCl,water, NaHCO3, brine, dried with MgSO4 and evaporated. 4.5 g crudeproduct was obtained (45%). ¹ H NMR: (CDCl₃, 200 MHz) d 7.80 (d, 2H,ArH), 7.33 (d, 2H, ArH), 4.15 (t, 2H, TsOCH₂), 3.80-3.45 (m, 10H, OCH₃),3.36 (d, 3H, OCH₃), 2.44 (s, 3H, ArCH₃).

EXAMPLE 54N-(3,6,9-Trioxadecyl)-5-Dimethoxytrityloxymethyl-3-Hydroxypyrrolidine

5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine (4.20 g, 10 mmol) and3,6,9-Trioxadecyl-4-Toluenesulfonate were dissolved in 30 ml THF, K₂ CO₃added (4.2 g, 30 mmol) and the suspension heated to reflux overnightwith stirring. A further 2.0 g 3,6,9-Trioxadecyl-4-Toluenesulfonate wasadded and the solution refluxed a further 4 hours. The solids werefiltered, the solvent evaporated and the product purified by flashchromatography using 1% TEA in EtOAc as eluant to give 4.18 g product(75%). ¹ H NMR: (CDCl₃, 200 MHz) d 7.45-6.80 (m, 13H, ArH), 4.35 (br m,1H, H3), 3.80-3.50 (m, 22H, OCH₃, H2a, b, H6a,b, OCH₂), 3.35 (d, 3H,OCH₃), 2.57 (br, 2H, NCH₃), 2.10-1.80 (bs, 2H, H4a,b).

EXAMPLE 55 4-Benzoyloxphenylacetic acid

Hydroxyphenylacetic acid (7.61 g, 50 mmol) and NaHCO₃ (10.1 g, 120 mmol)were dissolved in 250 ml water. The pH was adjusted to 9 with 2N NaOH.The solution was cooled to 0° C., and benzoyl chloride (6.40 ml, 7.73 g,55 mmol) added dropwise in 5 minutes. The Ph was maintained at 9 by theperiodic addition of 2N NaOH. After 20 min the ice bath was removed andstirring continued for 1 h. The solution was acidified to pH 2, and theprecipitate collected. 10.1 g product was obtained (79%).

EXAMPLE 56N-(4-Benzoyloxyphenylacetyl)-5-Dimethoxytrityloxymethyl-3-Hydroxypyrrolidine

To a stirred solution of 4-Benzoyloxyphenylacetic acid (3.84 g, 15.0mmol) and HOBT (3.45 g, 18.0 mmol) in 75 ml CH₂ Cl₂ was added EDC (2.43g, 18.0 mmol) and DIEA (3.2 ml, 2.3 g, 18 mmol). After 30 min,5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine (5.00 g, 11.9 mmol) wasadded in portions over 5 min, and stirred for 1 h. The reaction wasquenched after 1 h with 10 ml NaHCO₃, evaporated and the residueextracted twice with EtOAc, washed with NaHCO₃, brine, dried with MgSO4and evaporated. The product was purified by flash chromatography usingEtOAc/Hexane (4:1) as eluant to give 7.2 g product (92%). ¹ H NMR:(CDCl₃, 200 MHz) (2 rotamers) d 8.20-6.80 (22 H, ArH), 4.55, 4.45 (2m,1H, H3), 4.40, 4.22 (2m, 1H, H5), 3.77 (s, 6H, OCH₃), 3.65 (abq, 2H,COCH₂ Ar), 3.85, 3.50, 3.42, 3.20, 3.15 (4H, H2a, H2b, H6a, H6b), 2.15,1.90 (2m, 2H, H4a, H4b).

EXAMPLE 57 Nd-BOC-Guanidinoacetic acid

A suspension of Glycine ethyl ester hydrochloride (2.0 g, 14 mmol),N,N'-Bis-BOC-1-guanylpyrazole (Wu, Y., Matsueda, G. R., Bernatowicz, M.Synth. Commun. 1993, 23, 3055-60). (6.9 g, 22 mmol) and triethylamine(3.0 ml, 2.2 g, 21 mmol) were stirred vigorously in 120 ml DMF at RTovernight. Water was added and the solution was extracted twice withEtOAc, washed with 0.1N HCl, water, NaHCO₃, Brine, dried with MgSO₄ andevaporated. The residue was redissolved in 30 ml THF and 30 ml watercontaining 0.75 g NaOH was added, the mixture stirred vigorously for 4h. The basic solution was diluted with 75 ml water and washed twice with50 ml EtOAc. The aqueous layer was acidified to pH2, extracted twicewith EtOAc, washed with water, brine, dried with MgSO4 and evaporated togive pure product (2.69 g, 89%).

EXAMPLE 58N-(Nd-BOC-Guanidinoacetyl)-5-Dimethoxytrityloxymethyl-3-Hydroxypyrrolidine

To a stirred solution of Nd-BOC-Guanidinoacetic acid (2.69 g, 8.5 mmol)and HOBT (1.35 g, 10.0 mmol) in 75 ml CH₃ Cl₂ was added EDC (1.92 g,10.0 mmol) and DIEA (1.75 ml, 1.29 g, 10 mmol). After 30 min,5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine (3.2 g, 7.7 mmol}wasadded in portions over 5 min, and stirred for 1 h. The reaction wasquenched after 1 h with 10 ml NaHCO₃, evaporated and the residueextracted twice with EtOAc, washed with NaHCO₃, brine, dried with MgSO4and evaporated. The product was purified by flash chromatography using3% MeOH in CH₂ Cl₂ as eluant to give 4.4 g product (64%). ¹ H NMR:(CDCl₃, 200 MHz)(2 rotamers) d 7.42-6.80 (13 H, ArH), 4.58, 4.43 (1H, 2m, H3), 4.34, 4.12 (1H, 2 m, H5), 3.90, 3.70 (2H, 2 m, COCH₂ N), 3.72(6H, s, OCH₃), 3.55 (1H, m, H2a), 3.40 (2H, m, H2b, H6a), 3.15 (1H, d,H6b), 2, 28-1.90 (2H, 3 m, 2 H2), 1.40 (9 H, s, C(CH₃)₃).

EXAMPLE 59N-(3-Pyridylcarbonyl)-5-Dimethoxytrityloxymethyl-3-Hydroxypyrrolidine

A solution of nicotinoyl chloride hydrochloride (1.85 g, 10.5 mmol) in75 ml pyridine was added dropwise to a solution of5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine (4.20 g, 10 mmol) andtriethylamine (3.0 ml, 2.2 g, 21 mmol) in 50 ml pyridine. The solutionwas stirred at RT for 1 h, quenched with water (5 ml) and evaporated.The residue was extracted twice with EtOAc, washed with 0.1M citricacid, NaHCO3, brine, dried with MgSO4 and evaporated. The product wasobtained by flash chromatography using 3% MeOH+0.5% TEA in CH₂ Cl₂ aseluant. ¹ H NMR: (DMSO-d6, 200 MHz) d 8.65, 7.92, 7.52 (4H, Py--H),7.42-6.80 (13 H, ArH), 4.43 (1H, m, H3), 4.30 (1H, m, H5), 3.70 (7H, 2OCH₃, H2a), 3.30 (2H, m, H2b, H6a), 3.12 (dd, 1H, H6b), 2.10 (m, 2H,H4).

EXAMPLE 60N-(2-Acetamido-4-Methyl-5-Thiazolesulfonyl)-5-Dimethoxytrityloxymethyl-3-Hydroxypyrrolidine

2-Acetamido-4-Methyl-5-Thiazolesulfonyl chloride (956 mg, 3.95 mmol) wasdissolved in 50 ml CH₃ Cl₃ and 5 ml pyridine. This solution was addeddropwise to a solution of5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine (1.678 g, 4.00 mmol) andtriethylamine (0.83 ml, 0.61 g, 6.0 mmol) in 40 ml CH₂ Cl₂ at 0° C.After the addition was complete the solution was warmed to RT for 1 h,quenched with 10 ml water and evaporated. The reside was extracted withEtOAc, washed with 0.1M citric acid, NaHCO₃, brine, dried andevaporated. The product was purified by flash chromatography using 3%MeOH in CH₂ Cl₂ as eluant to give 1.57 g product (62%). ¹ H NMR: CDCl₃,200 MHz) d 7.42-6.80 (13 H, ArH), 4.42 (1H, m, H3), 4.07 (1H, m, H5),3.77 (6H, s, OCH₃), 3.70-3.25 (4H, m, 2H2, 2H6), 2.50 (3H, s, CH₃), 2.20(3H, s, COCH₃), 2.07 (2H, m, H4).

EXAMPLE 61N-(N',N'-Dimethylglycyl)-5-Dimethoxytrityloxymethyl-3-Hydroxypyrrolidine

To a stirred suspension of N,N-Dimethylglycine (1.24 g, 12.0 mmol) andHOBT (2.03 g, 15.0 mmol) in 75 ml CH₂ Cl₂ was added EDC (2.88 g, 15.0mmol) and DIEA (2.6 ml, 1.94 g, 15 mmol). After 30 min,5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine (4.61 g, 11.0 mmol) wasadded in portions over 5 min, and stirred. The reaction was quenchedafter 1 h with 10 ml NaHCO₃, evaporated and the residue extracted twicewith EtOAc, washed with NaHCO₃, brine, dried with MgSO4 and evaporated.4.15 g product was obtained (75%). ¹ H NMR: (CDCl₃, 200 MHz) d 7.42-6.80(13 H, ArH), 4.50 (2H, m, H3, H5), 3.80 (6H, s, OCH₃), 3.68 (2H, d,COH₂), 3.45 (1H, m, H2a), 3.15 (2H, m, H2b, H6a), 2.92 (1H, q, H6b),2.32, 2.24 (6H, 2 s, N(CH₃)₂), 2.15 (1H, m, H4a), 1.95 (1H, m, H4b).

EXAMPLE 62N-(Trifluoroacetyl)-5-Dimethoxytrityloxymethyl-3-Hydroxypyrrolidine

5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine (4.19 g, 10 mmol), ethyltrifluoroacetate (1.8 ml, 2.13 g, 15 mmol) and triethylamine (1 ml) werestirred in 50 ml methanol at RT overnight. The solvent was removed andthe residue purified by flash chromatography (2% MeOH/CH₂ Cl₂) to give4.92 g product (93%). ¹ H NMR: (CDCl₃, 200 MHz) d 7.42-6.80 (13 H, ArH),4.68 (1H, m, H5), 4.45 (1H, m, H3), 3.77 (8H, s, 2 OCH₃, 2 H2), 3.62(1H, q, H6a), 3.13 (2H, q, H6b ), 2.35 (1H m, H4a), 2.05 (1H, m, H4b).

EXAMPLE 63N-(3-Indoleacetyl)-5-Dimethoxytrityloxymethyl-3-Hydroxypyrrolidine

To a stirred solution of Indole-3-acetic acid (1.05 g, 6.0 mmol) andHOBT (0.95 g, 7.0 mmol) in 75 ml CH₂ Cl₂ was added EDC (1.34 g, 7.0mmol) and TEA (1.4 ml, 1.01 g, 10 mmol). After 30 min,5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine (2.1 g, 5.0 mmol) wasadded in portions over 5 min, and stirred for 1 h. The reaction wasquenched after 1 h with 10 ml NaHCO₃, evaporated and the residueextracted twice with EtOAc, washed with NaHCO₃, brine, dried with MgSO4and evaporated. The product was recrystallized from a minimal volume ofEtOAc to give 1.97 g product (68%). ¹ H NMR: (DMSO-d6, 200 MHz) d 10.85(s, 1H. indole-NH), 7.53-6.75 (17H, ArH), 4.35, 4.28 (2m, 1H, H3), 4.16(m, 1H, H5), 3.70 (s, 6H, OCH₃), 3.67 (abq, 2H, COCH₂), 3.47, 3.18,3.07, 2.96 (4m, 4H, H2a,b, H6a,b), 1.90, 1.80 (2m, 2H, H4a,b).

EXAMPLE 64N-(N-BOC-Glycyl)-5-Dimethoxytrityloxymethyl-3-Hydroxypyrrolidine

To a stirred solution of BOC-glycine (1.05 g, 6.0 mmol) and HOBT (0.95g, 7.0 mmol) in 75 ml CH₂ Cl₂ was added EDC (1.34 g, 7.0 mmol) and TEA(1.4 ml, 1.01 g, 10 mmol). After 30 min,5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine (2.1 g, 5.0 mmol) wasadded in portions over 5 min, and stirred for 1 h. The reaction wasquenched after 1 h with 10 ml NaHCO₃, evaporated and the residueextracted twice with EtOAc, washed with NaHCO₃, brine, dried with MgSO4and evaporated. The product was purified by flash chromatography (3%MeOH/CH₂ Cl₂) to give 2.68 g product (93%). ¹ H NMR: (DMSO-d6, 200 MHz)d 7.35-6.85 (13H, ArH), 6.70 (t, 1H, BOCNH), 4.40, 4.25 (2m, 1H, H3),4.16 (m, 1H, H5), 3.70 (s, 8H, COCH₂ N, OCH₃), 3.54, 3.27, 3.10, 3.00(4m, 4H, H2a,b, H6a,b), 1.95, 1.85 (2m, 2H, H4a,b), 1.38 (s, 9H, OtBu).

EXAMPLE 65 Phosphoramidite Synthesis: General Procedure

The alcohol, e.g. an N-substituted5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine (5.0 mmol) was dissolvedin 50 ml dry CH₃ CN and 2-cyanoethyl-N,N, N',N'-tetraisopropylphosphorodiamidite (1.9 ml, 6.0 mmol) added, followedby diisopropylammonium tetrazolide (340 mg, 2.0 mmol). The solution wasstirred until the alcohol was completely consumed (1-16 h), the solventevaporated, and the residue applied to a silica gel column. The productwas eluted with a mixture of EtOAc/Hexane containing 0.5% TEA.

EXAMPLE 66 H-phosphonate Synthesis: General procedure 1

Imidazole (6.81 g, 100 mmol) was dissolved in 400 ml dry CH₃ CN andcooled to 0° C. Phosphorus trichloride (2.62 ml, 4.12 g, 30 mmol) wasadded dropwise, followed by triethylamine (21 ml, 15.2 g, 150 mmol). Athick slurry developed to which was added over 15 min a solution ofalcohol, e.g. an N-substituted5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine (10 mmol) in 50 ml CH₃CN. Once the addition was complete, the ice bath was removed and thesolution stirred at RT for 30 min. The reaction was stopped by theaddition of 100 ml 1M triethylammonium bicarbonate (pH 7-8) buffer. Thesolvent was removed and the residue coevaporated with 100 ml Pyridine+5ml TEA, then 100 ml toluene. The residue was then dissolved in 0.05MTEAB., extracted (3×200 ml) with CH₂ Cl₂, and the extract washed with0.05M TEAB. The organic phase was dried with MgSO4 and concentratedunder reduced pressure. The product could be further purified by flashchromatography using a gradient of MeOH (1-10%) in CH₂ Cl₂ +1% TEA.

EXAMPLE 67 H-phosphonate Synthesis: General procedure 2

Phosphorus trichloride (1.3 ml, 2.06 g, 15 mmol) was added dropwise to asolution of triethylamine (21 ml, 15.2 g, 150 mmol) in 150 ml CH₂ Cl₂.1,2,4-Triazole (3.45 g, 50 mmol) was added, and the mixture cooled to 0°C. after 30 min. A solution of the alcohol, e.g. an N-substituted5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine (5.0 mmol) in 30 ml CH₂Cl₂ was added over 15 min. Once the addition was complete the solutionwas stirred for 30 min. The reaction was stopped by the addition of 100ml 1M triethylammonium bicarbonate (pH 7-8) buffer, the two phase systemstirred vigorously for 30 min. The product was extracted (3×200 ml) withCH₂ Cl₂, and the organic extract washed with 0.05M TEAB. The organicphase was dried with MgSO4 and concentrated under reduced pressure. Theproduct was purified by flash chromatography using a gradient of MeOH(1-10%) in CM₂ Cl₂ +1% TEA.

EXAMPLE 68 N-Palmitoyl-5-Dimethoxytrityloxymethylpyrrolidine-3-O-(N,N-Diisopropylamino)-2-Cyanoethoxyphosphite!

The title compound was prepared as per the procedure of Example 65 usingN-palmitoyl-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine of Example 5as the starting material.

EXAMPLE 69 N-Phenylacetyl-5-Dimethoxytrityloxymethylpyrrolidine-3-O-(N,N-Diisopropylamino)-2-Cyanoethoxyphosphite!

The title compound was prepared as per the procedure of Example 65 usingN-phenylacetyl-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine ofExample 7 as the starting material. ³¹ P NMR CDCl₃ δ 148.68, 148.26 and147.72 (two rotamers).

EXAMPLE 70N-(Fluorenylmethylsuccinoyl)-Dimethoxytrityloxymethylpyrrolidine-3-O-(N,N-Diisopropylamino)-2-Cyanoethoxyphosphite!

The title compound was prepared as per the procedure of Example 65 usingN-(fluorenylmethylsuccinoyl)-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidineof Example 9 as the starting material. ⁻ P NMR CDCl₃ δ148.6, 148.4,148.1, 148.0.

EXAMPLE 71 N-(N-1-Thymine)-2-acetyl!-5-Dimethoxytrityloxymethylpyrrolidine-3-O-(N,N-Diisopropylamino)-2-Cyanoethoxyphosphite!

The title compound was prepared as per the procedure of Example 65 usingN-(N-1-thymine)-2-acetyl!-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidineof Example 11 as the starting material.

EXAMPLE 72N-(N-Fmoc-3-Aminopropionoyl)-Dimethoxytrityloxymethyl-pyrrolidine-3-O-(N,N-Diisopropylamino)-2-Cyanoethoxyphosphite!

The title compound was prepared as per the procedure of Example 65 usingN¹-(N-Fmoc-3-Aminopropionoyl)-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidineof Example 13 as the starting material. ⁻ P NMR CDCl₃ δ 148.82, 148.61and 148.07 (two rotamers).

EXAMPLE 73N-(N-Imidazolyl-2-Acetyl)-Dimethoxytrityloxymethylpyrrolidine-3-O-(N,N-Diisopropylamino)-2-Cyanoethoxyphosphite!

The title compound was prepared as per the procedure of Example 65 usingN-(1-imidazolyl-2-acetyl)-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidineof Example 15 as the starting material. ³¹ P NMR CDCl₃ δ 149.2, 148.8,148.1 and 147.4 (two rotamers).

EXAMPLE 74 N-(Isobutyroyl)-Dimethoxytrityloxymethylpyrrolidine-3-O-(N,N-Diisopropylamino)-2-Cyanoethoxyphosphite!

The title compound was prepared as per the procedure of Example 65 usingN-(isobutyroyl)-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine ofExample 6 as the starting

material. ⁻ P NMR CDCl₃ δ 148.6, 148.0, 147.8 and 146.8 (two rotamers).

EXAMPLE 75 N-(N6-Benzoyl-9-Adenine)-2-Acetyl!-Dimethoxytrityloxymethylpyrrolidine-3-O-(N,N-Diisopropylamino)-2-Cyanoethoxyphosphite!

The title compound was prepared as per the procedure of Example 65 usingN-(N6-benzoyl-9-adeninyl)-2-acetyl!-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidineof Example 18 as the starting material.

EXAMPLE 76 N-(N4-Benzoyl-1-Cytosine)-2-Acetyl!-Dimethoxytrityloxymethylpyrrolidine-3-O-(N,N-Diisopropylamino)-2-Cyanoethoxyphosphite!

The title compound was prepared as per the procedure of Example 65 usingN-(N4-benzoyl-1-cytosine)-2-acetyl!-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidineof Example 21 as the starting material.

EXAMPLE 77 N-(N2-Isobutyroyl-9-Guanylyl)-2-Acetyl!-Dimethoxytrityloxymethylpyrrolidine-3-O-(N,N-Diisopropylamino)-2-Cyanoethoxyphosphite!

The title compound is prepared as per the procedure of Example 65 usingN-(N2-isobutyroyl-9-guanylyl)-2-acetyl!-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidineof Example 23 as the starting material.

EXAMPLE 78 N-(3, 6, 9,12-Tetraoxatridecanoyl)-5-Dimethoxytrityloxymethylpyrrolidine-3-O-(N,N-Diisopropylamino)-2-Cyanoethoxyphosphite!

The title compound is prepared as per the procedure of Example 65 usingN-(3,6,9,12-tetraoxatridecanoyl)-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidineof Example 26 as the starting material.

EXAMPLE 79 N-{Bis 2-(2-Pyridyl)ethyl!-Aminoacetyl}-Dimethoxytrityloxymethylpyrrolidine-3-O-(N,N-Diisopropylamino)-2-Cyanoethoxyphosphite!

The title compound is prepared as per the procedure of Example 65 usingN-{bis2-(2-pyridyl)ethyl!-aminoacetyl}-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidineof Example 29 as the starting material.

EXAMPLE 80N-(Trifluoromethanesulfonyl)-5-Dimethoxytrityloxymethyl-pyrrolidine-3-O-(N,N-Diisopropylamino)-2-Cyanoethoxyphosphite!

The title compound is prepared as per the procedure of Example 65 usingN-(trifluoromethanesulfonyl)-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidineof Example 31 as the starting material.

EXAMPLE 81 N-(Benzyl)-5-Dimethoxytrityloxymethylpyrrolidine-3-O-(N,N-Diisopropylamino)-2-Cyanoethoxyphosphite!

The title compound is prepared as per the procedure of Example 65 usingN-(benzyl)-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine of Example 32as the starting material.

EXAMPLE 82 N-(Aminocarbonyl)-5-Dimethoxytrityloxymethylpyrrolidine-3-O-(N,N-Diisopropylamino)-2-Cyanoethoxyphosphite!

The title compound is prepared as per the procedure of Example 65 usingN-(aminocarbonyl)-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine ofExample 33 as the starting material.

EXAMPLE 83N-(Methylaminothiocarbonyl)-2-Acetyl!-Dimethoxytrityloxymethylpyrrolidine-3-O-(N,N-Diisopropylamino)-2-Cyanoethoxyphosphite!

The title compound is prepared as per the procedure of Example 65 usingN-(methylaminothiocarbonyl)-2-acetyl!-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidineof Example 34 as the starting material.

EXAMPLE 84N-(Benzyloxycarbonyl)-5-Dimethoxytrityloxymethylpyrrolidine-3-O-(N,N-Diisopropylamino)-2-Cyanoethoxyphosphite!

The title compound is prepared as per the procedure of Example 65 usingN-(benzyloxycarbonyl)-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine ofExample 36 as the starting material.

EXAMPLE 85N-(N-α-Fmoc-α-Fluorenylmethyl-γ-glutamyl)-5-Dimethoxytrityloxymethylpyrrolidine-3-O-(N,N-Diisopropylamino)-2-Cyanoethoxyphosphite!

The title compound is prepared as per the procedure of Example 65 usingN-(N-α-Fmoc-α-fluorenylmethyl-δ-glutamyl)-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidineof Example 40 as the starting material.

EXAMPLE 86N-(Toluenesulfonyl)-5-Dimethoxytrityloxymethylpyrrolidine-3-O-(N,N-Diisopropylamino)-2-Cyanoethoxyphosphite!

The title compound is prepared as per the procedure of Example 65 usingN-(toluenesulfonyl)-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine ofExample 36 as the starting material.

EXAMPLE 87N-(N-Carbazolyl-2-Acetyl)-5-Dimethoxytrityloxymethylpyrrolidine-3-O-(N,N-Diisopropylamino)-2-Cyanoethoxyphosphite!

The title compound is prepared as per the procedure of Example 65 usingN-(N-Carbazolyl-2-Acetyl)-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidineof Example 42 as the starting material.

EXAMPLE 88N-(N-pyrrolyl-2-acetyl)-5-Dimethoxytrityloxymethylpyrrolidine-3-O-(N,N-Diisopropylamino)-2-Cyanoethoxyphosphite!

The title compound is prepared as per the procedure of Example 65 usingN-(N-pyrrolyl-2-acetyl)-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidineof Example 44 as the starting material.

EXAMPLE 89N-(2-Naphthylacetyl)-5-Dimethoxytrityloxymethylpyrrolidine-3-O-(N,N-Diisopropylamino)-2-Cyanoethoxyphosphite!

The title compound was prepared as per the procedure of Example 65 usingN-(2-naphthylacetyl)-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine ofExample 45 as the starting material. ³¹ P NMR CDCl₃ δ 148.5, 148.0,147.5.

EXAMPLE 90N-(Methanesulfonyl)-5-Dimethoxytrityloxymethylpyrrolidine-3-O-(N,N-Diisopropylamino)-2-Cyanoethoxyphosphite!

The title compound is prepared as per the procedure of Example 65 usingN-(methanesulfonyl)-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine ofExample 46 as the starting material. ³¹ P NMR CDCl₃ δ 149.2, 147.7.

EXAMPLE 91N-(N'-Trifluoroacetylglycyl)-5-Dimethoxytrityloxymethylpyrrolidine-3-O-(N,N-Diisopropylamino)-2-Cyanoethoxyphosphite!

The title compound is prepared as per the procedure of Example 65 usingN-(N'-trifluoroacetylglycyl)-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidineof Example 48 as the starting material. ³¹ P NMR CDCl₃ δ 149.0, 148.5.

EXAMPLE 92N-(1-Methylpyrrole-2-Carbonyl)-5-Dimethoxytrityloxymethylpyrrolidine-3-O-(N,N-Diisopropylamino)-2-Cyanoethoxyphosphite!

The title compound is prepared as per the procedure of Example 65 usingN-(1-methylpyrrole-2-carbonyl)-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidineof Example 52 as the starting material. ³¹ P NMR CDCl₃ α 148.2, 147.8.

EXAMPLE 93N-(3,6,9-Trioxadecyl)-5-Dimethoxytrityloxymethylpyrrolidine-3-O-(N,N-Diisopropylamino)-2-Cyanoethoxyphosphite!

The title compound is prepared as per the procedure of Example 65 usingN-(3,6,9-trioxadecyl)-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine ofExample 54 as the starting material.

EXAMPLE 94N-(4-Benzoyloxyphenylacetyl)-5-Dimethoxytrityloxymethylpyrrolidine-3-O-(N,N-Diisopropylamino)-2-Cyanoethoxyphosphite!

The title compound is prepared as per the procedure of Example 65 usingN-(4benzoyloxyphenylacetyl)-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidineof Example 56 as the starting material. ⁻ P NMR CDCl₃ δ 148.7, 148.4,148.2, 147.7.

EXAMPLE 95N-(Nd-BOC-Guanidinoacetyl)-5-Dimethoxytrityloxymethylpyrrolidine-3-O-(N,N-Diisopropylamino)-2-Cyanoethoxyphosphite!

The title compound is prepared as per the procedure of Example 65 usingN-(Nd-BOC-guanidinoacetyl)-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidineof Example 58 as the starting material.

EXAMPLE 96N-(3-Pyridylcarbonyl)-5-Dimethoxytrityloxymethylpyrrolidine-3-O-(N,N-Diisopropylamino)-2-Cyanoethoxyphosphite!

The title compound is prepared as per the procedure of Example 65 usingN-(3-pyridylcarbonyl)-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine ofExample 59 as the starting material.

EXAMPLE 97N-(2-Acetamido-4-Methyl-5-Thiazolesulfonyl)-5-Dimethoxytrityloxymethylpyrrolidine-3-O-(N,N-Diisopropylamino)-2-Cyanoethoxyphosphite!

The title compound is prepared as per the procedure of Example 65 usingN-(2-acetamido-4-methyl-5-thiazolesulfonyl)-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidineof Example 60 as the starting material.

EXAMPLE 98N-(N',N'-Dimethylglycyl)-5-Dimethoxytrityloxymethylpyrrolidine-3-O-(N,N-Diisopropylamino)-2-Cyanoethoxyphosphite!

The title compound is prepared as per the procedure of Example 65 usingN-(N|,N'-dimethylglycyl)-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidineof Example 61 as the starting material.

EXAMPLE 99N-(Trifluoroacetyl)-5-Dimethoxytrityloxymethylpyrrolidine-3-O-(N,N-Diisopropylamino)-2-Cyanoethoxyphosphite!

The title compound is prepared as per the procedure of Example 65 usingN-(trifluoroacetyl)-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine ofExample 62 as the starting material.

EXAMPLE 100N-(3-Indoleacetyl)-5-Dimethoxytrityloxymethylpyrrolidine-3-O-(N,N-Diisopropylamino)-2-Cyanoethoxyphosphite!

The title compound is prepared as per the procedure of Example 65 usingN-(3-indoleacetyl)-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine ofExample 63 as the starting material.

EXAMPLE 101 N-(N-BOC-Glycyl)-5-Dimethoxytrityloxymethylpyrrolidine-3-O-(N,N-Diisopropylamino)-2-Cyanoethoxyphosphite!

The title compound is prepared as per the procedure of Example 65 usingN-(N-BOC-Glycyl)-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine ofExample 64 as the starting material.

EXAMPLE 102N-(2-Naphthylacetyl)-5-Dimethoxytrityloxymethylpyrrolidine-3-O-HydrogenphosphonateTriethylammonium salt

The title compound is prepared as per the procedure of Example 65 usingN-(2-naphthylacetyl)-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine ofExample 45 as the starting material. ⁻ P NMR CDCl₃ δ3.77, J_(PH) =610Hz.

EXAMPLE 103N-(N'-Trifluoroacetyl)-5-Dimethoxytrityloxymethylpyrrolidine-3-O-HydrogenphosphonateTriethylammonium salt

The title compound is prepared as per the procedure of Example 65 usingN-(N'-trifluoroacetyl)-5-dimethoxytrityloxy methyl-3-hydroxypyrrolidineof Example 62 as the starting material. ⁻ P NMR CDCl₃ δ 3.85, J_(PH)=623 HZ.

EXAMPLE 104N-(N',N'-Dimethylglycyl)-5-Dimethoxytrityloxymethylpyrrolidine-3-O-HydrogenphosphonateTriethylammonium salt

The title compound is prepared as per the procedure of Example 67 usingN-(N',N'-dimethylglycyl)-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidineof Example 61 as the starting material. ⁻ P NMR CDCl₃ δ 3.77, J_(PH)=613 Hz.

EXAMPLE 105N-(Nd-BOC--Guanidinoacetyl)-5-Dimethoxytrityloxymethylpyrrolidine-3-O-HydrogenphosphonateTriethylammonium salt

The title compound is prepared as per the procedure of Example 67 usingN-(Nd-BOC-guanidinoacetyl)-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidineof Example 57 as the starting material. ⁻ P NMR CDCl₃ δ 3.82, J_(PH)=618.

EXAMPLE 106N-(4-Benzoyloxyphenylacetyl)-5-Dimethoxytrityloxymethylpyrrolidine-3-O-HydrogenphosphonateTriethylammonium salt

The title compound is prepared as per the procedure of Example 67 usingN-(4-Benzoyloxyphenylacetyl)-5-Dimethoxytrityloxymethyl-3-Hydroxypyrrolidineof Example 56 as the starting material. ⁻ P NMR CDCl₃ δ 3.86, J_(PH)=625 Hz.

EXAMPLE 107N-(2-Acetamido-4-Methyl-5-Thiazolesulfonyl)-5-Dimethoxytrityloxymethylpyrrolidine-3-O-HydrogenphosphonateTriethylammoniumsalt

The title compound is prepared as per the procedure of Example 67 usingN-(2-acetamido-4-methyl-5-thiazolesulfonyl)-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidineof Example 60 as the starting material. ⁻ P NMR CDCl₂ δ 3.77, J_(PH)=610.

EXAMPLE 108N-(3-Pyridylcarbonyl)-5-Dimethoxytrityloxymethylpyrrolidine-3-O-HydrogenphosphonateTriethylammonium salt

The title compound is prepared as per the procedure of Example 67 usingN-(3-pyridylcarbonyl)-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine ofExample 59 as the starting material. 31P NMR CDCl₃ δ 3.86, J_(PH) =624.

EXAMPLE 109 Synthesis of Hydrogenphoshponates

Using the procedure of Example 67 the following compounds are convertedto the DMT-hydrogenphosphonate from their respective DMT compoundspreviously synthesized above.

N-Isobutyroyl -5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine,N-palmitoyl -5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine,N-(phenylacetyl)-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine,N-(fluorenylmethylsuccinoyl)-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine,N- (N1-thymine)-2-acetyl!-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine, N-Fmoc-3-aminopropionoyl-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine,N-(1-imidazolyl-2-acetyl)-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine,N-(N-6-benzoyl-9aAdeninyl)-2-acetyl!-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine,N-(N-4-benzoyl-1-cytosine)-2-acetyl!-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine,N-(N2-isobutyroyl-9-guanine)-2-acetyl!-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine,N-3,6,9,12-tetraoxatridecanoyl!-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine,N- bis(2-(2-pyridyl)ethyl)-aminoacetyl!-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine,N-(toluenesulfonyl)-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine,N-(trifluoromethanesulfonyl)-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine,N-benzyl-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine,N-(aminocarbonyl)-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine ,N-(methylaminothiocarbonyl)-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine,N-(benzyloxycarbonyl)-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine,N-(N-α-Fmoc-α-fluorenylmethyl-δ-glutamyl)-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine,N-(N-carbanolyl-2-acetyl)-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine,N-(N-pyrrolyl-2-acetyl)-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine,N-(methanesulfonyl)-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine,N-(N'-trifluoroacetylglycyl)-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine,N-(1-methylpyrrole-2-carbonyl)-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine,N-(3,6,9-trioxadecyl)-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine,N-(3-indoleacetyl)-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine, andN-(N-BOC-glycyl)-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine.

EXAMPLE 110 Standard Oligomer Coupling Cycle Using Standard DNASynthesis Protocols

The oligomeric macromolecules of the invention are synthesized on anautomated DNA synthesizer (Applied Biosystems model 380B) as is donewith standard oligonucleotides using standard phosphoramidate chemistrywith oxidation by iodine (see, Oligonucleotide Synthesis, A PracticalApproach, M. J. Gait., ed., Oxford University Press, New York, 1990).For phosphorothioate oligomers, the standard oxidation bottle isreplaced by 0.2M solution of 3H-1,2-benzodithiole-3-one 1,1-dioxide inacetonitrile for the step wise thiation of the phosphite linkages. Thethiation wait step is increased to 68 sec and is followed by the cappingstep. After cleavage from the CPG column and deblocking in concentratedammonium hydroxide at 55° C. (18 hours), the oligomers are purified byprecipitation twice out of 0.5M NaCl solution with 2.5 volumes ethanol.Analytical gel electrophoresis is effected in 20% acrylamide, 8M urea,454 mM Tris-borate buffer, pH=7.0. Phosphodiester and phosphorothioateoligomers are judged from polyacrylamide gel electrophoresis as tomaterial length.

EXAMPLE 111 Synthesis of Sequence Specific Pyrrolidine Oligomer HavingPhosphodiester Linkages

"Aforvirsen" is an anti-papilloma agent having the nucleobase sequence:

TTG CTT CCA TCT TCC TCG TC.

A pyrrolidine phosphodiester linked oligomer of this preselectedsequence is prepared using the T, A, C and G reagents from Examples 71,75, 76 and 77, respectively, as per the procedure of Example 110 usingiodine as the oxidation reagent to give the phosphodiester linkedoligomeric compound having the "Aforvirsen" sequence.

EXAMPLE 112 Synthesis of Sequence Specific Pyrrolidine Oligomer HavingPhosphorothioate Linkages

A pyrrolidine phosphorothioate-linked oligomer of sequence TTG CTT CCATCT TCC TCG TC is prepared using the T, A, C and G reagents fromExamples 71, 75, 76 and 77, respectively, as per the procedure ofExample 110 using 3H-1,2-benzodithiole-3-one 1,1-dioxide as theoxidation reagent to give the phosphorothioate linked oligomericcompound.

EXAMPLE 113 Preparation of N-(2-1-Tyrosinyl!-acetyl)-2-hydroxymethylpyrrolidine-4-morpholinophosphoragmidate-thymidine-phosphodiester-thymidineTrimer.

A controlled pore glass resin derivatized with a 3'-5' phosphodiesterlinked dimer of thymidine 5'-Dmt--O--T--O--P(═O)--O--T--CPG! wassynthesized in a standard manner. Two aliquots of this resin (each 24.6mg, 47.5 mmole/g resin) were separately detritylated using 3%trichloroacetic acid in dichloromethane and then sequentially washedwith anhydrous acetonitrile and pyridine. Each was vigorously agitatedwith 1 mL of a solution of the N-(2-1-Tyrosinyl!acetyl)-2-O-dimethoxytrityloxymethyl-pyrrolidine-3-H-phosphonate monomer (25 mM) in adamantoyl chloride (50 mM) andpyridine for five and the mixture was shaken with the exclusion of lightfor 16 hrs. The mixture was filtered and washed with dichloromethane andthen diethylether (3×10 ml each). The CPG was shaken for 16 hrs inpyridine/water (4:1), filtered, and rinsed with pyridine (5×5 ml). A 10mg of the dried CPG was treated with 3% trichloroacetic acid indichloromethane. The presence of the trityl ion qualitatively verifiedthe derivatization. The loading was measured to be 30 μmol/g bymeasuring the absorbance of the dimethoxytrityl cation. minutes. Theresins were then washed repeatedly with anhydrous pyridine and blown drywith argon gas. To one column was added 1 mL of iodine/pyridine/water(2/90/8) solution and agitated vigorously; to the second column wasadded 1 mL of morpholine/carbon tetrachloride/pyridine (1/5/5) solutionand both were agitated for 30 minutes. The columns were subsequentlywashed with pyridine and acetonitrile and then blown dry with argon gas.Products were cleaved from the CPG by treatment with 1 mL ofconcentrated ammonium hydroxide for 30 minutes. The ammonia solutionswere evaporated to dryness and then treated with 80% acetic acid inwater for 30 minutes to remove the trityl group An aliquot of thesolution from the first column exhibited a single peak in an HPLCanalysis (27.8 min, Vyadec C-18, 260 nm, linear gradient of ammoniumacetate (pH 7)-acetonitrile, 0-75% acetonitrile in 52 minutes)corresponding to the phosphodiester trimer. A similar analysis yielded apair of HPLC peaks (39.7 and 39.9 min) corresponding to thephosphoramidate diastereomeric trimers.

In a parallel set of experiments using the HPP-Tyrosine H-phosphonatemonomer and the 5'-Dmt--O--T--O--P(═O)--O--T--CPG, the coupled resinswere oxidized to the phosphodiester trimer or to the phosphoramidatetrimer using dimethyl amine/carbon tetrachloride. These experimentsyielded the phosphodiester trimer and the unresolved dimethylaminephosphoramidate trimers at 12.4 min by an HPLC analysis, under theconditions described before.

EXAMPLE 114 Derivatization of LCAA CPG With2-O-(Dimethoxytrityl)-ethylsuccinate Half Ester

2-O-(Dimethoxytrityl)ethylsuccinate half ester triethylammonium salt(135 mg) was dissolved in dichloromethane (5 ml).4-Dimethylaminopyridine catalyst (40 mg, 0.2 mmol) was added followed bytoluene diisocyanate (0.029 ml, 0.2 mmol). The mixture was shaken for 18min. Long chain alkyl amine controlled pore glass (LCAA CPG) (1.0 g) wasadded

EXAMPLE 115 Hydrogen phosphonate Coupling: General Procedure.¹

A portion of solid support (CPG or other polymeric support e. g.TentaGel) derivatized with a DMT protected alcohol linked via asuccinate linker (1 μmol) was loaded into a DNA synthesis column, andattached to an automated DNA synthesizer programmed to perform thefollowing functions.

1) Wash with dichloromethane

2) DMT group was removed with 3% trichloroacetic acid indichloromethane.

3) Wash with dichloromethane and CH₃ CN/Pyridine (1:1).

4) Coupling: addition of alternating portions of 0.2M Adamantoylchloride or Pivaloyl chloride in CH₃ CN/Py (1:1) and 0.05M H-phosphonatemonomer in CH₃ CN/Py (1:1) for 1 min.

5) Wash with CH₃ CN/Pyridine (1:1).

6) Repeat steps 1 through 5 until the full sequence is completed.

The product of this sequence of reactions is an H-phosphonate diesterwhich is oxidized by one of several methods including those describedbelow.

Oxidation Procedure 1: Phosphodiester

The solid support-bound H-phosphonate diester is treated (manually orautomatically) with equal volumes of solution A (0.2M I₂ in THF) andsolution B (N-methylmorpholine/H₂ O/THF 1:1:8) for 5 min, followed byequal volumes of solution A and solution C (TEA/H₂ O/THF 1:1:8) for 5min, followed by washing with CH₃ CN/Py (1:1).

Oxidation Procedure 2: Phosphorothioate

The solid support-bound H-phosphonate diester is treated (manually orautomatically) with a solution of S₈ in CS₂ /Lutidine for 30 min. Thesolid support is then washed with CH₃ CN/Py (1:1).

Oxidation Procedure 3: Phosphoramidate

The solid Support-bound H-phosphonate diester is treated (manually orautomatically) with a solution of the required amine (10% V/V) in CCl₄4/Pyridine 1:1 for 15-30 min. The solid support is then washed with CH₃CN/Py (1:1).

Synthesis of Phosphoramidate Library with Uniform PN-Substitution,X_(PN) (N_(PN))_(n) N

The library is synthesized on Ethylene glycol CPG or on UniversalSupport CPG (Zeneca, Cambridge, UK) using an automated DNA synthesizer.The solid support is separated into a number of portions equal to thenumber of monomers used. Each monomer is coupled to the support usingthe H-phosphonate coupling general procedure. The portions are mixed andredivided for the next coupling. In this way, the random oligomers aresynthesized until the fixed position is reached. The support is againsplit into portions and each monomer coupled. Additional monomers can beadded after the fixed position by performing the couplings to theindividual pools. Additional random positions can be added to theoligomer by further splitting each of the pools and recombining aftercoupling, taking care not to mix pools with different fixed positions.Once the oligomer assembly is complete, the H-phosphonate linkages inthe individual subsets need to be oxidized, following general procedure3. This has the result of producing the same phosphoramidate linkagebetween each residue in the library. By following oxidation generalprocedure 1 or 2 instead, an all phosphodiester or all phosphorothioatelibrary is produced. The library pools are then cleaved from the solidsupport, purified and assayed for activity as described previously.

Synthesis of Phosphoramidate Library with fixed PN-Substitution, X_(PN1)-N_(PN2) -(N_(PNn))_(m) -N

Synthesis of a phosphoramidate library usingDMT-hydroxyprolinol-H-phosphonate monomers (P-Amidate: HPP, Variableamines, X_(PN1) -N_(PN2) -(N_(PNn))_(m) -N). The library is synthesizedon Ethylene glycol CPG or on Universal Support CPG (Zeneca, Cambridge,UK) using an automated DNA synthesizer. The solid support is separatedinto a number of portions equal to the number of monomers used (X). Eachmonomer is coupled to the support using the H-phosphonate couplinggeneral procedure. The portions are recombined, and the H-phosphonatediester linkage is converted to a phosphoramidate using Oxidationprocedure 3. These steps are repeated (split, couple, combine), followedby Oxidation procedure 3 using a different amine for each randomizedposition in the oligomer. At the fixed position, the support is split,the monomers are coupled, but the subsets are kept separate for theoxidation step. After cleavage and workup, one obtains a library withrandomized monomers and a different substituent at each phosphoramidatelinkage. It is also possible to introduce phosphodiester andphosphorothioate linkages at any position in the oligomer library bysubstituting the appropriate oxidation procedure.

Synthesis of Phosphoramidate Library with Variable PN-Substitution,X_(PNn) -(N_(PNn))_(m) -N

The library is synthesized on Ethylene glycol CPG or on UniversalSupport CPG (Zeneca, Cambridge, UK) using an automated DNA synthesizer.The solid support is separated into a number of portions equal to thenumber of monomers used. Each monomer is coupled to the support usingthe H-phosphonate coupling general procedure. The portions arerecombined, mixed and separated into portions. Each portion is oxidizedwith a different amine following oxidation procedure 3, followed bycombining and mixing of the supports. These steps (split, couple, mix,split, oxidize, mix) are repeated are repeated until the fixed position.The support is then split into portions, the monomer coupled, and theresin portions split again for oxidation following procedure 3. Afterworkup, each subset has a unique monomer residue and phosphoramidatelinkage, and an equimolar amount of each monomer and amine in therandomized portion. It is also possible to incorporate phosphodiesterand phosphoramidate linkages randomly into the oligomers by oxidizing aportion of each H-phosphonate diester by following procedure 1 or 2.

EXAMPLE 116 Incorporation of Monomeric Units Into Oligomeric StructureUsing Combinatorial Technique--General Procedure

A solid support (universal support from Cambridge Research Biochemicals)is separated into portions of equal weight. The number of portionsequals the number of monomers in the combinatorial library. Each portionis reacted with a desired amidites using tetrazole as catalyst, followedby oxidation of the phosphite triester to the phosphate as per thestandard coupling cycle of Example 110 above. The DMT ether is cleavedusing trichloroacetic acid in CH₂ Cl₂ regenerate the hydroxyl group atthe end of the extended oligomer. The extent of the coupling reaction isoptimized to be ≧90% completed by varying the amidite concentration andtotal equivalents and the coupling time. After a coupling, the supportis mixed thoroughly, then divided equally and amidites are again reactedindividually to a portion of the support. This cycle is repeated foreach random position until the `fixed` position is reached.

At the `fixed` position of the oligomer, each amidite is reactedindividually to a portion of the support, but the portions are notmixed. Instead, each subset is further divided into the number ofportions corresponding to the number of monomers. Each portion ofsupport is then reacted with a different amidite, followed by mixing asabove. Repeating this cycle for each of the different subsets ofsupports results in randomization in positions following the fixedposition in the sequence. The resulting subsets are unique only in thefixed position.

At completion of the oligomer synthesis, the oligomers are cleaved fromthe solid support and phosphate protecting groups are removed byincubation for 1-2 hours at room temperature in concentrated ammonia.Supernatant containing the oligomer is then removed from the silica andincubated at 55° C. for 6-16 hours to cleave the protecting groups fromthe residues. The oligomer is desalted and protecting groups are removedby HPLC size exclusion chromatography.

EXAMPLE 117 Evaluation of Coupling Efficiency of PhosphoramiditeMonomers

The following method is used to evaluate the hydroxyprolinolphosphoramidites or other phosphoramidites for suitability of use in arandom sequence solid state oligomer synthesis. A solid-phase synthesissupport containing an internal reference is used to determine couplingefficiency, estimate the extinction coefficient, and evaluatecoupling-product quality of the test phosphoramidite monomers asfollows:

A test monomer-support is selected as is an internal standard. Using dTas a symbol of thymidine, dC as a symbol for deoxy cytidine and otherabbreviations as note in the text below, in an illustrative test system,thymidine bound to CPG, identified as dT-CPG, is used for the testmonomer-support and 5'-O-acetyl capped cytosine bound to CPG, identifiedas 5'-Ac-dC-CPG, is used for the internal standard.

Reactive dT-CPG is mixed with a lesser molar equivalent of unreactive5'-Ac-dC-CPG. The unreactive 5'-Ac-dC-CPG internal standard allows foraccurate determination of unreacted dT present before and after acoupling reaction.

The peak area of dT can be identified as A_(T) and the peak area of dCidentified as A_(C). The initial ratio of peak areas for dT and dC,i.e., (A_(T) /A_(C))₀, is determined by cleavage, deprotection, and HPLCanalysis of an aliquot of the CPG mixture. Measurements are taken at awavelength of 260 nm. Relative moles of dC can be identified as C, andrelative moles of dT can be identified as T. These are calculated frompeak areas, A_(C) and A_(T), respectively, using known extinctioncoefficients: C=A_(C) /ε_(C) and T=A_(T) /ε_(T). Thus the relative peakarea or molar amount of dT initially present can always be calculatedfrom the peak area of dC:

A_(T0) =(A_(C)) (A_(T) /A_(C))₀ !

T₀ =(C) (T/C)₀ !

also,

(C)(T/C)=(A_(C) /ε_(C)) (A_(T) /ε_(T))/(A_(C) /ε_(C))!=(A_(C)/ε_(C))(A_(T) /A_(C)))(ε_(C) ε_(T))

thus,

(C) (T/C)₀ !=(A_(C) /ε_(C))) (A_(T) /A_(C))₀ !(ε_(C), ε_(T)) =(A_(C)/ε_(T)) (A_(T) /A_(C))₀ !

An amidite monomer of interest, identified as X, is reacted with analiquot of the CPG mixture. Reacted CPG is cleaved and deprotected withammonia, then analyzed by HPLC to determine the area under the peak fordC, i.e., A_(C) ; area under the peak for unreacted dT, i.e., A_(Tur) ;and area under the peak for X-T dimer, i.e., A_(XT). These values areused to calculate coupling efficiency, C.E.; and X-T dimer extinctioncoefficient ε_(XT).

The coupling efficiency, C.E., is defined by the ratio of reacted dT,i.e., T_(r), to total dT, i.e., T₀. Thus C.E.=T_(r) /T₀. Couplingefficiency can be determined from the relative moles of unreacted dTpresent before, i.e., T₀, and after, i.e., T_(ur), coupling with X; allthree are related by the equation

    T.sub.0 =T.sub.r +T.sub.ur.

Since C.E. is a unit-less value, HPLC peak areas can be used instead ofrelative molar quantities to perform the calculation:

C.E.=(T_(r) /T₀) =(T₀ /T₀)-(T_(ur) /T₀) =1-(T_(ur) /T₀) =1-(A_(Tur)/ε_(T))(A_(T0) /ε_(T)) =1-(A_(Tur) /A_(T0)) =1-(A_(Tur))(A_(C)) (A_(T)/A_(C))₀ !!

The foregoing are all measurable quantities.

The extinction coefficient ε for X, i.e., ε_(X-T), in the given HPLCsolvent system is determined from the C.E. for X and the relative areasof the HPLC peaks. The amount of X-T is equal to the amount of T thathas reacted. ε for dimer X-T is defined as the peak area A_(XT) dividedby the moles of X-T dimer present XT, and is calculated as follows:

XT=T_(r) =(C.E.)(T₀)

ε_(XT) =(A_(XT) /XT) =(A_(XT))/(C.E.)(T₀) =(A_(XT))/(C.E.)(C) (T/C)₀!=(A_(XT))/(C.E.)(A_(C) /ε_(T)) (A_(T) /A_(C))₀ !

These, again, are all measurable quantities.

Finally, the quality of the coupling-product X-T can be evaluated fromthe appearance of the HPLC chromatogram. Significant peaks (thosesumming>10% of product-peak area) other than those expected might alsobe addressed. Often they are the desired X-T dimer that retainsprotective groups. Disappearance of these peaks with extended ammoniatreatment will confirm that the monomer requires extended ammoniadeprotection beyond the standard time. In other cases the extra peakscan be identified as undesirable side-products or in some case theycannot be identified. Generally, coupling efficiency of less than about90%, a required ammonia deprotection time of greater than a few days, orthe occurrence of side-products amounting to greater that 10% (by UVabsorbance) can be selected as initial guidelines to judge thepossibility of excluding an amidite from consideration for use in aparticular set of amidites used in generating random oligomericcompounds.

EXAMPLE 118 Evaluation of Coupling Efficiency of IllustrativePyrrolidine Phosphoramidite Monomer

The phenylacetylhydroxyprolinol phosphoramidite of Example 69 wascoupled to a dT-CPG solid-phase synthesis support forming dimers andtrimers. Coupling was effected as per the general procedure of Example110. Synthesis of the dimers and trimers was performed with an ABI 394DNA synthesizer (Applied Biosystems, Foster City, Calif.) using standardDNA synthesis reagents and synthesis protocols, with the exception of anextended (5 minute) coupling time added to the synthesis cycle. Theoligomers were cleaved from solid support by treatment with concentratedammonia for 3 days at 4° C. The supernatant was removed from the supportand heated in a sealed vial at 55° C. for eight hours. This solution wascooled, and most of the ammonia removed by evaporation. The oligomerswere analyzed directly on reversed-phase HPLC column (Waters Nova-PakPhenyl, cat. #10656; Millipore Corp., Milford, Mass.) using a gradientof 1% to 75% acetonitrile in 0.1M M ammonium acetate, pH 7, over 50minutes. The HPLC system was a Waters with a 991 detector, 625 LC pump,and 714 WISP auto-injector. Calculations were performed using datacollected at a wavelength of 260 nm.

In an iteration of this procedure, a dimer of the benzylhydroxyprolinolcoupled to dT was synthesized as described above with the terminal DMTgroup removed to give a free terminal hydroxyl. The resulting crudemixture was analyzed by HPLC. Identification of all but one of the HPLCchromatogram peaks was made. The coupling efficiency and extinctioncoefficient of the dimer was determined using the general proceduredescribed in Example 110, above, as follows:

C.E.=1-(A_(Tur))/(A_(C)) (A_(T) /A_(C))₀ !=1-(7.20)/(16.16)(5.57)=0.92(92%)

ε_(XT) =(A_(XT))/(C.E.)(A_(C) /ε_(T)) (A_(T) /A_(C))₀!=(68.34)/(0.92)(7.20/8.71)(5.57) =16.1M⁻¹ cm⁻¹ (10⁻³)

The quality of the product was thus within a preselected ≧90% limit. Theunaccounted for "impurity" material, represented only about 5% of thetotal area of the product peaks.

EXAMPLE 119 PLA₂ assay

Phospholipase A2 (PLA2) enzymes are responsible for the hydrolysis ofthe sn-2 linkage of membrane phospholipids. PLA₂ -catalyzed reaction isthe rate-limiting step in the release of a number of pro-inflammatorymediators, and type II PLA2 is implicated in the pathogenesis of severalhuman inflammatory diseases. Library subsets were screened forinhibition of the activity of type II PLA2 and a unique inhibitor wasidentified.

The oligomer libraries were screened for inhibition of PLA₂ in an assayusing E. coli cells labeled with ³ H-oleic acid as the substrate.Franson et al., J. Lipid Res. 1974, 15, 380; and Davidson et al., J.Biol. Chem. 1987, 262, 1698. Type II PLA₂ (originally isolated fromsynovial fluid), expressed in a baculovirus system and partiallypurified, serves as a source of the enzyme. A series of dilutions (inwater) of each of the oligomeric pools was made: 10 μL of each oligomerwas incubated for 5 minutes at room temperature with a mixture of 10 μLof PLA₂, 20 μL 5×PLA₂, buffer (500 mM Tris, pH 7.0-7.5, 5 mM CaCl2) and50 μL water. Each of the oligomer samples was run in duplicate. At thispoint, 10 μL of ³ H-labeled E. coli cells was added. This mixture wasincubated at 37° C. for 15 minutes. The enzymatic reaction was haltedwith the addition of 50 μL of 2M HCl and 50 μL of fatty acid-free BSA(20 mg/mL PBS), vortexed for 5 seconds and centrifuged at high speed for5 minutes. A 165 μL portion of each supernatant was then put into ascintillation vial containing 6 mL of scintillant (Scintiverse) and cpmswere measured in a Beckman Liquid Scintillation Counter. As a control, areaction without oligomer was run alongside the other reactions as wellas a baseline reaction containing neither oligomer nor PLA₂ enzyme. Cpmswere corrected for by subtracting the baseline from each reaction datapoint. The results are shown in Table III.

                  TABLE III                                                       ______________________________________                                        Round  Sequence*      Most Active X=                                                                             IC.sub.50 (μM)                          1      XNNNNdT        egOC         40                                         2      (egOC)XNNNdT   egOC         5                                          3      (egOC) (egOC)XNNdT                                                                           egOC         1.5                                        ______________________________________                                    

*N represents an equimolar mixture of egIM, egOC, egNH, hpPH, hpSU andhpNY, wherein hpPH isN-Phenylacetyl-5-Dimethoxytrityloxymethylpyrrolidine-3-O-(N,N-diisopropylamino)-2-cyanoethoxyphosphite!, hpSU isN-(fluorenylmethylsuccinoyl)-dimethoxytrityloxymethylpyrrolidine-3-O-(N,N-diisopropylamino)-2-cyanoethoxyphosphite!, and hpNY is theunsubstituted hydroxyprolinol phosphoramidite.

The first round selected (egOC)NNNNdT as the most active subset with anIC₅₀ of 40 μM. The next round led to the determination of the monomerunit in the second position of the oligomer, i.e. egOC, with aneight-fold improvement in activity and an IC₅₀ of 5 μM. The third roundof synthesis and selection led to the identification of (egOC)(egOC)-(egOC)NNdT as the most active subset with a still furtherimproved IC₅₀ of 1.5 μM. Further rounds can be used to identify a uniqueoligomer with the greatest activity in the PLA₂ assay.

EXAMPLE 120 Verification of Assay

The PLA₂ test system of Example 119 as verified using phosphorothioateoligonucleotides with one or more strings of guanosine nucleotides (atleast 3 Per string). Libraries of these oligonucleotides weredeconvoluted using the SURFs screening strategy and were shown to havean inhibitory effect on the PLA₂ enzyme. Knowing that phosphorothioateoligonucleotides inhibit PLA₂ with some sequence specificity, an eightnucleotide phosphorothioate library consisting of the four natural baseswas used to test the assay system for suitability as a SURF screen. Thislibrary had been synthesized for use in another system and all subsetswere not still available (indicated by dashes in Table IV, below). Usingthe SURF method, it was confirmed that a stretch of guanosines werenecessary for inhibition of PLA₂ activity by the phosphorothioateoligonucleotide (Table IV, below).

The assay was sensitive and accurate enough to discriminate betweensubsets of oligomers so that an inhibitory sequence could be selected.In each of the first three rounds of selection, the most active subsetwas readily determined. After 5 rounds, there was little difference inthe activity of the subsets with at least 5 G's in a row, suggestingthat the terminal positions are not critical for the inhibitoryactivity. The IC₅₀ of the "winner" improves (enzyme activity decreases)as more of the positions are fixed. As a test of the reproducibility ofthe assay, an eight nucleotide phosphorothioate oligonucleotide of asingle sequence (TTGGGGTT) was assayed with each round of testing. Thisoligonucleotide acted as an internal control of the accuracy of theassay; the IC₅₀ was 8 μM in each assay.

                  TABLE IV                                                        ______________________________________                                        Inhibition of PLA.sub.2 Activity by Library                                   Subsets IC.sub.50 (mM)                                                                   X=A  X=G        X=C    X=T                                         ______________________________________                                        Round 2                                                                       NNGNXNNN     >50    25         >50  >50                                       Round 3                                                                       NNGXGNNN     --     10         >50  --                                        Round 4                                                                       NNGGGXNN     9      4          6    18                                        Round 5                                                                       NAGGGGXN     4      2          4    4                                         NGGGGGXN     2.5    2          3    3                                         NCGGGGXN     5      4          5    5                                         NTGGGGXN     19     5          17   15                                        ______________________________________                                    

EXAMPLE 121 Assay of Library of Pyrrolidine Oligomeric Compounds AgainstPLA₂

A library containing the novel prolinol monomers is tested in the PLA₂assay for identification of inhibitors of type II PLA₂. Confirmation ofthe "winner" is made to confirm that the oligomer binds to enzyme ratherthan substrate and that the inhibition of any oligomer selected isspecific for type II PLA₂. An assay using ¹⁴ C-phosphatidyl ethanolamine(¹⁴ C-PE) as substrate, rather than E. coli membrane, is used to insureenzyme rather than substrate specificity. Micelles of ¹⁴ C-PE anddeoxycholate are incubated with the enzyme and oligomer. ¹⁴ C-labeledarachidonic acid released as a result of PLA₂ -catalyzed hydrolysis isseparated from substrate by thin layer chromatography and theradioactive product is quantitated. The "winner" is compared tophosphatidyl ethanolamine, the preferred substrate of human type IIPLA₂, to confirm its activity. PLA₂ from other sources (snake venom,pancreatic, bee venom) and phospholipase C, phospholipase D andlysophospholipase can be used to further confirm that the inhibition isspecific for human type II PLA₂.

EXAMPLE 122 Hybridization probe for the detection of specific mRNA inbiological sample

For the reliable, rapid, simultaneous quantification of multiplevarieties of mRNA in a biological sample without the need to purify themRNA from other cellular components, a mRNA of interest from a suitablebiological sample, i.e., mRNA of a blood borne virus, a bacterialpathogen product in stool, urine and other like biological samples, isidentified using standard microbiological techniques. An oligomericcompound of the invention complementary to the nucleic acid sequence ofthis mRNA is prepared as per the above examples. The oligomeric compoundis immobilized on insoluble CPG solid support utilizing the procedure ofPon, R. T., Protocols for Oligonucleotides and Analogs, Agrawal, S.,Ed., Humana Press, Totowa, N.J., 1993, p 465-496. Using the method ofPCT application WO 93/15221, a known aliquot of the biological sampleunder investigation is incubated with the insoluble CPG support havingthe oligomer thereon for a time sufficient to hybridize the mRNA tooligomer and thus to link the mRNA via the oligomer to the solidsupport. This immobilizes mRNA present in the sample to the CPG support.Other non-immobilized materials and components are then washed off theCPG with a wash media suitable for use with the biological sample. ThemRNA on the support is labelled with ethidium bromide, biotin or acommercial radionucleotide and the amount of label immobilized on theCPG support is measured to indicate the amount of mRNA present in thebiological sample.

Those skilled in the art will appreciate that numerous changes andmodifications may be made to the preferred embodiments of the inventionand that such changes and modifications may be made without departingfrom the spirit of the invention. It is therefore intended that theappended claims cover all such equivalent variations as fall within thetrue spirit and scope of the invention.

EXAMPLE 123 Leukotriene B₄ assay

Leukotriene B₄ (LTB₄) has been implicated in a variety of humaninflammatory diseases, and its pharmacological effects are mediated viaits interaction with specific surface cell receptors. Library subsetswere screened for competitive inhibition of radiolabeled LTB₄ binding toa receptor preparation.

A Nenquest™ Drug Discovery System Kit (NEN Research Products, Boston,Mass.) was used to select an inhibitor of the interaction of LeukotrieneB₄ (LTB₄) with receptors on a preparation of guinea pig spleen membrane.³ H! Leukotriene B₄ reagent was prepared by adding 5 mL of liganddiluent (phosphate buffer containing NaCl, MgCl₂, EDTA and Bacitracin,pH 7.2) to 0.25 mL of the radioligand. The receptor preparation was madeby thawing the concentrate, adding 35 mL of ligand diluent and swirlinggently in order to resuspend the receptor homogenously. Reagents werekept on ice during the course of the experiment, and the remainingportions were stored at -20° C.

The library subsets were diluted to 5 μM, 50 μM and 500 μM in phosphatebuffer (1×PBS, 0.1% azide and 0.1% BSA, pH 7.2), yielding final testconcentrations of 0.5 μM, 5 μM and 50 μM, respectively. Samples wereassayed in duplicate. ³ H! LTB₄ (25 μL ) was added to 25 μL of eitherappropriately diluted standard (unlabeled LTB₄) or library subset. Thereceptor suspension (0.2 mL) was added to each tube. Samples wereincubated at 4° C. for 2 hours. Controls included ³ H! LTB₄ withoutreceptor suspension (total count vials), and sample of ligand andreceptor without library molecules (standard).

After the incubation period, the samples were filtered through GF/Bpaper that had been previously rinsed with cold saline. The contents ofeach tube were aspirated onto the filter paper to remove unbound ligandfrom the membrane preparation, and the tubes washed (2×4 mL) with coldsaline. The filter paper was removed from the filtration unit and thefilter disks were placed in appropriate vials for scintillationcounting. Fluor was added, and the vials shaken and allowed to stand atroom temperature for 2 to 3 hours prior to counting. The counts/minute(cpm) obtained for each sample were subtracted from those obtained fromthe total count vials to determine the net cpm for each sample. Thedegree of inhibition of binding for each library subset was determinedrelative to the standard (sample of ligand and receptor without librarymolecules). The results are shown in Tables V.

                  TABLE V                                                         ______________________________________                                        Round  Sequence*      Most Active X=                                                                             IC.sub.50 (μM)                          ______________________________________                                        1      XNNNNdT        egOC         15                                         2      (egOC) XNNNdT  egOC         7                                          3      (egOC) (egOC)XNNdT                                                                           egIM         2                                          ______________________________________                                         *N represents an equimolar mixture of egOC, egIM, egNH, hpPH,                 hpSU and hpNY.                                                           

The first two rounds of synthesis and selection were useful in thedetermination of the first two monomer units of the oligomer, i.e.(egOC)(egOC)NNNdT, with an IC₅₀ of 7 μM. The next round led to theselection of an oligomer with an improved IC₅₀ of 2 μM. Further roundscan be performed for the identification of a unique oligomer with thebest activity.

We claim:
 1. A compound having structure II: ##STR3## wherein: X is H, a phosphate group, an activated phosphate group, an activated phosphite group, a solid support, a conjugate group, or an oligonucleotide;Y is H, a hydroxyl protecting group, a conjugate group or an oligonucleotide; E is O or S; EE is O³¹, or N(Y₀)T₀ ; Y₀ is H, or Q₂ !_(jj) --Z₂ ; T₀ is Q₁ !_(kk) --Z₁, or together Y₀ and T₀ are joined in a nitrogen heterocycle; Q₁ and Q₂ independently, are C₂ -C₁₀ alkyl, C₂ -C₁₀ alkenyl, C₂ -C₁₀ alkynyl, C₄ -C₇ carbocylo alkyl or alkenyl, a heterocycle, an ether having 2 to 10 carbon atoms and 1 to 4 oxygen or sulfur atoms, a polyalkyl glycol, or C₇ -C₁₄ aralkyl; jj and kk independently, are 0 or 1; Z₁ and Z₂, independently, are H, C₁ -C₂₀ alkyl, C₂ -C₂₀ alkenyl, C_(2-C) ₂₀ alkynyl, C₆ -C₁₄ aryl, C_(7-C) ₁₅ aralkyl, a halogen, CH═O, OR₁, SR₂, NR₃ R₄, C(═NH)NR₃ R₄, CH(NR₃ R₄), NHC(═NH) NR₃ R₄, CH(NH₂)C(═O)OH, C(═O)NR₃ R₄, C(═O)OR₅, a metal coordination group, a reporter group, a nitrogen-containing heterocycle, a purine, a pyrimidine, a phosphate group, a polyether group, or a polyethylene glycol group; Z is alkyl having 1 to about 20 carbon atoms, alkenyl having 2 to about 20 carbon atoms, alkynyl having 2 to about 20 carbon atoms, aryl having 6 to about 20 carbon atoms, OR₁, SR₂, NR₃ R₄, C(═NH)NR₃ R₄, NHC(═NH)NR₃ R₄, CH═O, C(═O)OR₅, CH(NR₃ R₄) (C(═O)OR₅), C(═O)NR₃ R₄, OH, SH, SCH₃, NR₃ R₄, NR₃ R₄, a nitrogen-containing heretocycle, a purine, a pyrimidine, a phosphate group, a polyether group, a polyethylene glycol group or metal coordination groups; R₁ is H, alkyl having 1 to about 6 carbon atoms, or a hydroxyl protecting group; R₂ is H, alkyl having 1 to about 6 carbon atoms, or a thiol protecting group; R₃, and R₄, are, independently, H, alkyl having 1 to about 6 carbon atoms, or an amine protecting group; R₅ is H, alkyl having 1 to about 6 carbon atoms, or an acid protecting group; Q is alkyl having 1 to about 6 carbon atoms, acyl having 1 to about 6 carbon atoms, C(O)--O, C(O)--NH, C(S)--O, C(S)--NH, or S(O)₂ ; n is 0 or 1; and m is 1 to about
 50. 2. A chimeric oligomeric compound having a central region comprising a phosphodiester or a phosphorothioate oligodeoxynucleotide interspaced between flanking regions having structure II: ##STR4## wherein: X is H, a phosphate group, an activated phosphate group, an activated phosphite group, a solid support, a conjugate group, or an oligonucleotide;Y is H, a hydroxyl protecting group, a conjugate group or an oligonucleotide; E is O or S; EE is 0⁻, or N(Y₀)T₀ ; Y₀ is H, or Q₂ !_(jj) --Z₂ ; T₀ is Q₁ !_(kk) --Z₁, or together Y₀ and T₀ are joined in a nitrogen heterocycle; Q₁ and Q₂ independently, are C₂ -C₁₀ alkyl, C₂ -C₁₀ alkenyl, C₂ -C₁₀ alkynyl, C₄ -C₇ carbocylo alkyl or alkenyl, a heterocycle, an ether having 2 to 10 carbon atoms and 1 to 4 oxygen or sulfur atoms, a polyalkyl glycol, or C₇ -C₁₄ aralkyl; jj and kk independently, are 0 or 1; Z₁ and Z₂, independently, are H, C₁ -C₂₀ alkyl, C₃ -C₂₀ alkenyl, C₂ -C₂₀ alkynyl, C₆ -C₁₄ aryl, C₇ -C₁₅ aralkyl, a halogen, CH═O, OR₁, SR₂, NR₃ R₄, C(═NH)NR₃ R₄ CH (NR₃ R₄), NHC(═NH) NR₃, R₄, CH(NH₂) C(═O)OH, C(═O)NR₃ R₄, C(═O)OR₅, a metal coordination group, a reporter group, a nitrogen-containing heterocycle, a purine, a pyrimidine, a phosphate group, a polyether group, or a polyethylene glycol group; Z is alkyl having 1 to about 20 carbon atoms, alkenyl having 2 to about 20 carbon atoms, alkynyl having 2 to about 20 carbon atoms, aryl having 6 to about 20 carbon atoms, OR₁, SR₂, NR₃ R₄, C(═NH)NR₃ R₄, NHC(═NH)NR₃ R₄, CH═O, C(═O)OR₅, CH(NR₃ R₄) (C(═O)OR₅), C(═O)NR₃ R₄, OH, SH, SCH₃, NR₃ R₄, NR₃ R₄, a nitrogen-containing heterocycle, a purine, a pyrimidine, a phosphate group, a polyether group, a polyethylene glycol group or metal coordination groups; R₁ is H, alkyl having 1 to about 6 carbon atoms, or a hydroxyl protecting group; R₂ is H, alkyl having 1 to about 6 carbon atoms, or a thiol protecting group; R₃ and R₄ are, independently, H, alkyl having 1 to about 6 carbon atoms, or an amine protecting group; R₅ is H, alkyl having 1 to about 6 carbon atoms, or an acid protecting group; Q is alkyl having 1 to about 6 carbon atoms, acyl having 1 to about 6 carbon atoms, C(O)--O, C(O)--NH, C(S)--O, C(S)--NH, or S(O)₂ ; n is 0 or 1; and m is 1 to about
 50. 3. The compound of claim 1 wherein Y is a hydroxyl protecting group.
 4. The compound of claim 3 wherein Y is trityl, methoxytrityl, dimethoxytrityl or trimethoxytrityl.
 5. The compound of claim 1 wherein X is H, an activated phosphite group, or a solid support.
 6. The compound of claim 5 wherein X is a phosphoramidite.
 7. The compound of claim 1 wherein n is 1 and Q is acyl and is carbonyl, thiocarbonyl, carboxy, acetyl or succinyl.
 8. The compound of claim 1 wherein Z is a nitrogen-containing heterocycle.
 9. The compound of claim 8 wherein said nitrogen-containing heterocycle is imidazole, pyrrole or carbazole.
 10. The compound of claim 9 wherein Z is imidazole.
 11. The compound of claim 1 wherein Z is a purine or a pyrimidine.
 12. The compound of claim 11 wherein Z is adenine, guanine, cytosine, uridine or thymine.
 13. The compound of claim 12 wherein Q is acetyl.
 14. The compound of claim 1 wherein Z is alkyl having 1 to about 20 carbon atoms.
 15. The compound of claim 1 wherein Z is aryl having 6 to about 14 carbon atoms or aralkyl having 7 to about 15 carbon atoms.
 16. The compound of claim 15 wherein Z is fluorenylmethyl, phenyl, or benzyl.
 17. The compound of claim 1 wherein Z is polyethylene glycol or glutamyl.
 18. The compound of claim 1 wherein m is 1 to about
 25. 19. The compound of claim 1 wherein E is O.
 20. The compound of claim 1 wherein E is S.
 21. The compound of claim 1 wherein said Z groups of are in a predetermined sequence.
 22. The compound of claim 1 wherein said Z groups of are in a random sequence.
 23. A synthetic process comprising:selecting a group of monomers having structure I: ##STR5## wherein: X is H, a phosphate group, an activated phosphate group, an activated phosphite group, or a solid support; Y is H or a hydroxyl protecting group; Z is alkyl having 1 to about 20 carbon atoms, alkyl having 2 to about 20 carbon atoms, alkynyl having 2 to about 20 carbon atoms, aryl having 6 to about 20 carbon atoms, OR₁, SR₂, NR₃ R₄, C(═NH)NR₃ R₄, NHC(═NH)NR₃ R₄, CH═O, C(═O)OR₅, CH(NR₃ R₄)(C(═O)OR₅), C(═O)NR₃ R₄, OH, SH, SCH₃, NR₃ R₄, NR₃ R₄, a nitrogen-containing heterocycle, a purine, a pyrimidine, a phosphate group, a polyether group, a polyethylene glycol group or metal coordination groups; R₁ is H, alkyl having 1 to about 6 carbon atoms, or a hydroxyl protecting group; R₂ is H, alkyl having 1 to about 6 carbon atoms, or a thiol protecting group; R₃ and R₄ are, independently, H, alkyl having 1 to about 6 carbon atoms, or an amine protecting group; R₅ is H, alkyl having 1 to about 6 carbon atoms, or an acid protecting group; Q is alkyl having 1 to about 6 carbon atoms, acyl having 1 to about 6 carbon atoms, C(O)--O, C(O)--NH, C(S)--O, C(S)--NH, or S(O)₂ ; and n is 0 or 1; and covalently bonding at least two monomers of said group to form said compound.
 24. The process of claim 23 wherein the Z moiety of at least one monomer of said group is different from the Z moiety of another monomer of said group.
 25. The process of claim 23 wherein said randomized oligomeric compound includes from 2 to 50 of said monomers.
 26. An oligomeric compound prepared by the process of claim 23 wherein said compound includes from 2 to 25 of said monomers. 